Molecular mechanisms underlying Campylobacter jejuni's unusual swimming style

Campylobacter jejuni is a bacterium that causes food poisoning. In the UK Campylobacter causes more food poisoning than other bacteria such as E. coli or Salmonella (e.g, the Food Standards Agency estimates that Campylobacter infections cost us almost one billion pounds per year). Campylobacter is also very similar to other '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 proposal uses Campylobacter as an example to understand swimming in this family of dangerous bacteria.

Most dangerous bacteria need to be able to swim to cause their disease, and Campylobacter swims in a very unusual manner. Most 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 the motor spins; the spinning long tail curls up to become a helical propeller, pushing the bacterium through its liquid habitat. Campylobacter (and family members) 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 be able to develop targeted drugs that only affect Campylobacter and family. Specifically, Campylobacter uses a single very powerful motor to swim. It also uses this powerful motor to rotate its body, which is shaped like a corkscrew, allowing it to 'bore' into very thick fluids such as gut mucous easily, and therefore is better able to cause disease. But we don't understand how Campylobacter is able to do many of these things. 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's really important to be able to see the motor and the shapes of the cell, which is a difficult task. I believe this is absolutely essential, so to visualize bacteria I trained in a technique that enables us to directly see them inside the cell, and the molecular details of the motor that drives swimming. To help me fully understand the images from this project I've recruited a stellar team of international scientists.

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.

I propose four aims:

One: Campylobacter actually makes two motors, but only uses one of them (if it used both, they'd push in opposite directions!). I'll use my imaging skills to see both the active and inactive motors to understand what the difference is - in turn, telling us how it works. If we can understand how one motor is inactivated, maybe we can make drugs to inactivate both motors?

Two: Campylobacter swims by rotating both its tail, and its cell body. I think I know how it divides the amount of swimming power between the two, and will test this.

Three: How does Campylobacter make itself helical-shaped? I'll collect images to make progress towards understanding this.

Four: Finally, I'll team up with collaborators to develop a mathematical model to combine all previous results to understand how all factors combine to produce the unique swimming of Campylobacter.

The aim of this research is to 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 and collaborations in genetic manipulation, cell biology, crystallography, and biophysics, with the ultimate goal of developing a quantitative model of this swimming. 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 drug development in the era of antimicrobial resistance.

All electron cryo-tomographic data will be collected using established protocols that have already been published.

This proposal describes four aims. Aims 1 to 3 are fairly independent; aim 4 integrates results into a model:

Aim 1: Use electron cryo-tomography to determine the structures of active and inactive motors in C. jejuni, identify the protein(s) responsible for inactivation, and locate those protein(s) in the structures. Methods will be developed to separate active and inactive motors for structure determination. Differences between structures of the wild-type motor, and motors with genes deleted or tagged will enable location of proteins.

Aim 2: Use electron cryo-tomography, genetics, and modelling, to test the hypothesis that the C. jejuni flagellar 'hook' that couples the flagellum to the cell body has evolved to alter the ratio of rotation (and therefore thrust) between the flagellum and counter-rotation of the helical cell body.

Aim 3: Use electron cryo-tomography and genetics to make headway into understanding how C. jejuni makes itself into a helical shape, essential for proper swimming.

Aim 4: Use fluorescent labelling of C. jejuni and develop mathematical models to fully understand C. jejuni with an integrative model.

This project has considerable relevance to the strategic priorities of the MRC. Outcomes will be basic discoveries in a prime area for drug design to tackle antimicrobial resistance, conforming to MRC Strategic Aim 1 ("Picking research that delivers") (and furthermore evidenced by my track record). I also outline a number of public outreach activities that meet with MRC Strategic Aim 2 ("Research to people": "Communication"). This research is also particularly relevant to Strategic Aim 3 ("Going Global"), as C. jejuni is a worldwide problem, and results will be directly relevant to closely-related Helicobacter and Arcobacter species. Collaborating with the Hendrixson, Nishizaka, Samatey, Salama, and Poon labs conforms to Strategic Aim 3's "Partnerships and shaping the agenda" as all are international leaders in their respective fields, thus confering a significant competitive advantage. Furthermore, Strategic Aim 4's "Supporting scientists": "Capacity and skills" is met by this proposal: In 2012 I was in a position to stay in the US to launch my independent career (and am well situated to return should I choose); MRC funding will enable me to consolidate a strong international lab. Furthermore this project meets Strategic Aim 4's "Research Environment" in terms of supporting a high-throughput electron cryo-tomography lab in the UK which will be able to provide expert advice to other labs interested in the technology.

The following people beyond immediate academic peers may benefit from this research:

Industry. Results from this research may culminate in industrial collaborations, patenting, or foundation of a start-up company to develop novel antimicrobials. This benefit will be realized through Imperial Innovations, the Imperial College London technology commercialization company.

Basic life scientists. The microbiology and C. jejuni communities will learn about the structure, mechanisms, and evolution of molecular machinery. This information will be communicated by myself and the unnamed postdoctoral research assistant through peer-reviewed publications, and invited and informal talks at conferences and on university campuses.

Evolutionary biologists. A profound question in biology lies in how complexity develops, and how proteins have come together over the course of evolution to form large macromolecular machines. This project will form the basis for understanding how this might happen. Again, this information will be presented to the scientific community through talks and presentations.

The public. I am promoting and publicizing my work to the public in a number of ways. I exhibit yearly at the Imperial Festival to directly present my work, and engage with the Natural History Museum (our first museum stall will be hosted at the end of September at an evening event). I am also engaging with a number of artist colleagues to disseminate concepts on biological self-assembly and self-organization. Furthermore I actively publicize results; my recent 2016 PNAS paper was highlighted in (amongst others) New Scientist, Gizmodo.com, The Daily Mail, and a three-page feature article in Science et Vie (a French popular science magazine with circulation of ~300 000). Despite being international, all work will prominently feature the Imperial and MRC brand identities. All of these projects will synergistically serve to focus my communication abilities with a non-specialist audience.

Students. As with non-scientists, students are excited to learn about molecular machinery, particularly when the methods used for study incorporate new, exciting methods such as electron cryo-tomography. I already incorporate this research (biology and technique) into lectures to inspire undergraduates, and will continue to do so. PhD students who I will mentor will also benefit from this cutting-edge approach and biology.