The bacterial type IV pilus machinery as a DNA translocator

Bacteria are single-celled organisms that are surrounded by membranes made of lipids. Membranes maintain the shape and structure of the cell and also act as a semi-permeable barrier, allowing the exchange of small nutrients and waste products, but preventing leakage of larger molecules like DNA and proteins. However, many molecules need to cross membranes in order to reach the destination where they perform their function, which may be in DNA replication, protein synthesis or even as part of the "engine" of the cell, which uses energy to turn a motor and drive bacterial movement.

To cross membrane barriers, many different systems exist. The overarching theme of my research is the study of a large and powerful protein complex called the type IV pilus (T4P) assembly machinery, which performs two completely different functions. The first is transporting small protein subunits across the cell membrane to produce an extremely long filament (a pilus) on the cell surface. The pilus is very dynamic and can be assembled and retracted extremely quickly, enabling cells to "walk" across surfaces in a jerky manner. The pilus also acts as a means of communication between cells, and can be a key factor in enabling bacteria to cause disease. In our previous work, we have identified two different forms of pilus that are assembled from the same machine, but it is not clear how they are related to bacterial behaviour. It is imperative that we further our knowledge of pili by conducting fundamental biosciences research. In this way, mechanisms that prevent bacterial movement and colonisation can be developed by more applied research strategies in the future.

The second function of some T4P machines is their ability to support DNA uptake from the extracellular environment. It is not entirely clear if this also involves the pilus filament in some form. Uptake of DNA is called "natural competence" and can be an extremely dangerous phenomenon as it enables cells to "feed" on foreign DNA and incorporate it into their own genome. This means that previously non-risk species can behave unpredictably and become pathogenic to animals, crops and humans. By the same mechanism, bacteria can develop antimicrobial resistance, which is a massive worldwide healthcare problem. We plan to develop an assay involving fluorescent labelling, that will enable DNA binding and uptake to be monitored in whole cells, which in the subsequent part of this work will lead on to identifying the specific binding site and proteins involved. This is an extremely important aspect of research that must be conducted in order to better understand the underlying mechanisms that bacteria use to take up genetic material.

The main technique that will be employed in this research is electron cryo-tomography (cryoET). This is a state-of-the-art approach, which employs an electron microscope in order to visualise whole cells and determine protein structures, which is extremely powerful in the burgeoning sphere of structural cell biology. Using cryoET in this study will allow us to visualise the entire T4P machinery performing different functions in bacterial cells. The work will therefore uncover new information to further our understanding of a fundamental molecular machine, and how it performs a dual-function in nature.

Many bacterial species express type IV pili (T4P) on their surface, which determine cell virulence, enable motility, communication and biofilm formation. Commonly, the T4P assembly machinery is also able to take up DNA from the extracellular environment via a process referred to as "natural competence". Sharing genetic information in this way enables genome plasticity, the development of pathogenicity and resistance to antimicrobials. Understanding the T4P machinery at a fundamental level is thus a key prerequisite in fathoming the evolution of antimicrobial resistance and how this is manifested at the level of the organism.

We aim to characterise the T4P machinery as a dual-function system, by delineating the structural basis for the two mechanisms employing techniques centred on cryoEM and cryoET. Despite the central importance of natural competence, it is not clear how the mechanism works, or if and to what extent T4P are involved. We will develop a new method to monitor DNA binding and uptake with the aim of determining the specific protein components involved. We will complement this by determining high-resolution structures of two previously identified T4P forms and investigating their ability to bind and take up DNA. This will be correlated to their protein composition by mass spectrometry, and to their tensile strength and assembly by atomic force microscopy. Taken together, this information will be used to create models of the entire T4P assembly machinery and DNA translocator in situ, enabling the implications of both processes to be interpreted in the context of the entire system.

Homologues of the T4P machinery are also involved in other secretory pathways (type II and type III secretion systems), which help bacteria to infect, manipulate and kill their hosts. Thus, our system will provide valuable new information which can ultimately be used to exploit findings in biotechnology and medicine towards the development of new antimicrobial treatments.

By employing a multi-disciplinary approach with a focus on state-of-the-art cryoEM, the immediate aim of this work is to understand a fundamental biological mechanism occurring at bacterial cell membranes. The immediate impact will lie in scientific advancement and the generation of new knowledge, pertinent to our understanding of membrane protein transporters, bacterial motility, communication and natural competence. Understanding mechanisms of both cell motility and natural competence will further our knowledge of how bacteria exploit our environment and undergo genetic adaptation, both of which are important processes that contribute to the current global healthcare problem of AMR. This research is therefore extremely timely with respect to the UK governments research priority (2013-2018) to underpin the dynamics of AMR transmission.

We will explore the T4P machinery as a dual-function system by determining protein structures and developing a method to label DNA for cryoEM. This will encourage other researchers to apply similar methodology in the study of other transporters or membrane protein machines. CryoEM will be conducted in the new South West Regional Facility for CryoEM, in collaboration with other researchers within GW4 (Exeter, Bristol, Bath and Cardiff). Technical knowledge gained will be bestowed to other users through steering committee meetings and courses aimed at training other scientists, in order to promote multi-lateral communication and sharing of ideas within the UK scientific community. Data will be submitted to the EM data bank for use by other researchers globally, academics and industry alike. The pharmaceutical industry will benefit from structural knowledge of the DNA transporter, one example being in the design of targeted drugs to prevent DNA uptake or cell motility. Knowledge gained of bacterial movement and colonisation will be of interest to industrial partners and biotechnology companies in the development of surfaces that are incompatible with colony formation. This would revolutionise the ability to insert catheters and perform implant surgeries. Such products would be extremely beneficial for the population and also lucrative for the economy. Mechanisms are in place at Exeter to develop and exploit potential commercial opportunities (see Pathways to Impact).

The project will also generate a trained PDRA with highly desirable expertise in protein biochemistry and cryoEM. This researcher will be encouraged to further their professional development through courses and workshops. They will also be encouraged to assist with transmission of research within the academic community and beyond. We will not only disseminate research through the standard routes (publications in international journals and presentations at conferences), but also firmly commit to engagement of wider audiences. Particularly important findings will be communicated via press releases and twitter feeds, and we will encourage budding young scientists to engage with us through the Big Bang South West Fair. Imaging research can be used to develop interactive scientific art, providing an attractive and interesting way to communicate complex science to the public, and above all exemplifying the importance of publicly funded research. We will also promote our research and technology to scientists working in other disciplines, for example at the Exeter Café Scientifique and Pint of Science festival, fostering the development of new collaborations and ideas. By championing our research, I will aim to encourage a broader uptake in biophysics, particularly for female scientists. By acting as a successful female role model, I will encourage future generations of researchers by inviting school-level and university students to tours of our facility, showcasing fun and interactive ways of displaying and understanding scientific data, and encouraging them to pursue a high-value future in the field of research and development.