Sequential assembly of the bacterial flagellum outside the living cell

Bacteria are the smallest of all cells and live lives of feast and famine. To survive they must sense and adapt quickly to their ever-changing external environment. To find food and escape danger, many bacteria build on their cell surface long rotating propellers called flagella, which allow them to move rapidly over surfaces and through liquids. Motility driven by surface flagella is important to colonization of rich niches in human, animal and plant hosts by disease-causing (pathogenic) bacteria. Both biologists and physicists have long found flagella fascinating as they illustrate beautifully how complex biological structures, comprising a number of discrete substructures, are assembled meticulously from thousands of distinct protein subunit building blocks to operate as tiny rotary 'nanomachines' outside the living cell. They have even been claimed by creationists, who cite their exquisite structural-functional complexity as evidence against slow evolution!

Remarkably, the flagellum is fundamentally self-assembling and this feat of nanoscale construction is rendered even more astonishing by the fact that each new subunit building block, made inside the cell, must travel up to 20 times the length of the cell through a narrow central channel in the growing flagellum to incorporate at its tip far outside the cell. Until recently, the mystery was how the subunit building blocks travel through the long channeI where there is no discernable energy source to propel them. We have discovered that this is powered by the subunits themselves, as they link in a chain that is pulled to the flagellum tip.

We now propose to use molecular, biochemical and structural biology approaches to build on this completely new and unanticipated chain mechanism for flagellum growth to address a major open question concerning the assembly of the complex flagellum structure: how are the many different subunit building blocks incorporated into the flagellum in the correct sequence? The proposed work has the potential to reveal new aspects of flagellum assembly and provide a deeper understanding of how multi-component biological machines can be assembled beyond the surface of living cells. Findings from this work could, ultimately, be exploited to discover new molecules to block the function of such machines, leading to the development of new drug therapies to treat bacterial infections.

Flagella, the helical propellers that extend from the bacterial cell surface, are a paradigm for how complex molecular machines are built outside the living cell. Their sequential assembly requires export of thousands of structural subunits across the cell membrane and this is achieved by a type III export machine located at the flagellum base. Subunits then transit up to 20 cell lengths through a narrow channel in the flagellum to reach the assembly site at the flagellum tip. We have recently solved the mystery of how subunit transit is achieved without a conventional energy source in the external channel, showing that the energy for transit is intrinsic to the subunits themselves as they link in a chain pulled to the flagellum tip (Nature 504:287).

Our proposal builds on this new chain mechanism and preparatory experiments arising from it, and is primed to reveal serial key mechanisms for subunit recognition at the export machinery, sequential subunit assembly and length-control in the flagellum. We will use our biochemical approaches to determine whether subunit binding affinities determine the order in which subunits link into the chain and incorporate into the nascent flagellum. We will build on initial NMR and crystallography to gain a structural view of subunit interactions with the export gate, revealing binding interfaces and conformational changes triggered by subunit binding. Finally, we will assess interactions at the export gate and with subunit chains by the molecular ruler FliK, which monitors hook length and governs the export switch from rod/hook to filament subunits, an approach that could uncover the mechanism by which FliK reports hook length back to the export gate, a critical and unexplained event in the sequential assembly of flagella. In addition to resolving central features of flagella assembly, our work will be pertinent to understanding how bacterial virulence needles are built, and indeed to the wider subject of export and assembly.

Private sector beneficiaries: While the immediate impact of our research will be in the academic community, in the longer term our work has potential for impact in the pharmaceutical, biotechnology and nanotechnology industries. Firstly, our work will potentially uncover new targets for novel antimicrobials that block bacterial motility or effector export. Secondly, protein export via the flagellar pathway has already become a focus for biotechnologists working to develop systems for rapid and simple purification of large amounts of recombinant proteins from the extracellular growth media (e.g. Nature Biotechnology 23:475-81). Our proposed work has the potential to reveal new aspects of flagellar export that could be exploited to further improve the secretion of heterologous proteins via the flagellar pathway. Lastly, as detailed in the academic beneficiaries section, our work on flagella export has already attracted attention in the field of nanotechnology and our work could have implications for the self-assembly of multicomponent nanotubes. Should we reach the point where aspects of our research show strong potential for translation to industry, we will take appropriate steps (see Pathways to Impact) and work with Cambridge Enterprise to engage with industry, patent discoveries, and translate our research into commercially exploitable resources.

We take very seriously the need to train and develop new cohorts of researchers skilled in current molecular microbiology, structural biology and biochemical techniques that are vital to the continued success of UK science and to maintain UK competitive advantage in the increasingly knowledge-led economy, especially in the face of increasing microbiological challenges of the future. Such researchers are in high demand in the industrial sector, and previous members of our labs have taken leading positions in UK industry. In addition to acquiring valuable technical expertise, the RA employed on this project will work with the PI, Co-I, Cambridge University Personal and Professional Development and the University Careers Service to develop transferable professional skills (e.g. Leadership and Management Development, Presentation and Communication) that will equip them for transition to many employment sectors.

Beneficiaries in the wider public : We anticipate a number of impacts. In the short-to-medium term, there will be benefits from our planned engagement activities (see Pathways to Impact). Part of the fight to combat infectious disease lies in generating public awareness: by learning about research like ours the public become aware of the science behind the global health threat that will be posed in the future by re-emergent resistant bacteria. Such an understanding is important in seeking to augment research support. A major aspect of our public engagement effort will be schools outreach work. We view this as essential to foster interest in STEM subjects. The PI, Co-I and RA employed on the project will regularly engage students (e.g. through Nuffield scholarships), giving us further opportunity to inspire the next generation of researchers.

Longer-term benefits to the public will emerge should our work translate to industry. Flagella and related needle structures are important virulence factors in a wide range of human, animal and plant infections. Treatment by existing antibiotics is threatened by rising resistance so fundamental research into virulence mechanisms is vital to our understanding of infections, and for our ability to develop new interventions. Ultimately, the target beneficiaries are the millions suffering from bacterial infections and our hope is that such work will facilitate design of new drugs that could attenuate bacterial motility and effector export and be used as adjuncts to antibiotic-based therapies. In this case, the work will also impact upon those developing new antimicrobial therapies both in academia and in pharmaceutical research.