Unraveling type IV pilus assembly

Pili are hair-like appendages found in many different bacteria that allow them to "stick" to many different surfaces. In bacteria causing disease in humans, they often promote attachment to host cells and colonization. No pili are more widespread than type IV pili (Tfp) that are found in hundreds of bacterial species, including many that cause life-threatening diseases such as Neisseria meningitidis (responsible for meningitis and septicaemia) and Vibrio cholerae (responsible for cholera).

Studying pilus biology is crucial because a better understanding of the way in which bacteria assemble these filaments is expected to provide leads for the design of new drugs. Interfering with pilus assembly would prevent colonization and thus "disarm" pathogenic bacteria. This is a promising alternative to the massive use of antibiotics that has resulted in an unrelenting spread of antibiotic resistance, which is a major public health concern worldwide. There is now experimental evidence for some very well characterized pili that this approach can work. However, our understanding of how Tfp are assembled is far too limited for such an approach and needs to be improved, which is the main objective of the research proposed here.

Tfp biogenesis relies on a very complex machinery of up to 18 highly conserved proteins. We have provided indirect evidence that Tfp assembly relies on a relatively small subset of these proteins. Our recent finding that this machinery can self-assemble in the bacterial model par excellence Escherichia coli paves the way for a "Lego" approach in which a minimal Tfp assembly machinery would be reconstituted in E. coli through sequential addition of the required meningococcal components.

The two aims of this research project are therefore to:

1. reconstitute in E. coli a minimal system that is capable of assembling Tfp through modular co-expression of N. meningitidis pil genes,

2. perform and in depth functional characterization of the Tfp assembly machinery in order to better understand its mode of action.

This is expected to have a major impact on Tfp biology and be key for the future design of drugs capable of interfering with Tfp assembly, with are expected to have an exceptionally wide spectrum of action.

Pili are hair-like appendages found in many bacterial species to which they confer adhesive abilities. In numerous human pathogens they promote attachment to host cells and thereby play a key role in pathogenesis. Type IV pili (Tfp) are undoubtedly the most widespread type of pili since they are found in hundreds of very different bacterial species, including many important human pathogens such as our model Neisseria meningitidis.

As already demonstrated for better characterized pili, understanding the molecular basis of Tfp assembly is expected to provide leads for the rational design of small molecules interfering with that process. Due to Tfp's exceptionally widespread distribution, such molecules would have a large spectrum of action and be of a tremendous therapeutic potential. However, this will need our understanding of Tfp assembly to be dramatically improved, which is the main objective of the research proposed here.

Using a systematic genetic approach in N. meningitidis, we have provided indirect evidence that Tfp assembly relies on a relatively small set of highly conserved proteins, many of which were found to form a sub-complex at the inner membrane. Our recent finding that this sub-complex can self-assemble upon expression of the corresponding genes in E. coli paves the way for the reconstitution of a minimal system for Tfp assembly in the bacterial "workhorse" par excellence. Reconstituting a minimal pilus assembly system in E. coli will allow its subsequent functional and structural characterization, which are the main objectives of this research project. This is expected to have a major impact on Tfp biology.

The two aims of this research project are to:

1. reconstitute in E. coli a minimal system capable of assembling Tfp through modular co-expression of optimized synthetic N. meningitidis pil genes,

2. perform and in depth functional characterization of this machinery in order to better understand its mode of action.

The outcome of this research project will be the elucidation of the molecular mechanisms of Tfp assembly. Due to the widespread distibution of Tfp in prokaryotes, and their key role in virulence in numerous human pathogens, this is likely to have practical implications by providing leads for the future design of novel therapeutic approaches with a wide spectrum of action. The following beneficiaries have been identified and some methods of how we will ensure that they have the opportunity to benefit from this research are described.

1. Academia
Our findings will have significant impact on the very broad academic community studying Tfp and other related fundamental biological processes. We will ensure that results of this research are disseminated widely by Open Access publication in high-impact journals and presentation at international research conferences (often prior to publication).

2. Pharma and UK industry
This research will lay the foundation for the design of selective inhibitors of Tfp assembly, which is envisioned beyond this research project. This is a very attractive alternative to antibiotics for the development of new therapies against bacterial pathogens that place a heavy burden on human health and economy, and is as such expected to be of interest to small and big Pharma companies. We will, when appropriate, ensure that intellectual property opportunities are maintained through liaison via MRC and ICL technology transfer expertise (IC INNOVATIONS Ltd). Our local expertise will advise on how to protect inventions and develop an appropriate IP protection strategy. Furthermore, business and translational offices at ICL will help foster relationships with industry partners. Where appropriate they might co-invest in new to accelerate development and increase value.

The PDRA and any undergraduate, postgraduate, or part-time students that will contribute to the project will develop key interdisciplinary skills that will be extremely valuable for UK industry and contribute to the knowledge economy and increase the economic competitiveness of the UK. The PDRA will be trained in key techniques and good laboratory practice, encouraging innovative approaches to research. There are also opportunities for training and supervision of undergraduate, postgraduate, or intern students at ICL. Much of the preliminary work in this application has already contributed towards the training of undergraduate and postgraduate students. The PDRA will be encouraged to present his/her work as widely as possible, and to communicate with both short-term and long-term user groups. Training in communication will be provided. ICL has a highly active staff development programme, which includes courses on presenting science to a lay audience, developing an independent research career, grant writing and exploiting translational aspects of research.

3. Public sector healthcare professionals
Although the outcomes will not impact directly on healthcare during the course of the grant, the research is expected to contribute in the future to drug design and so we will engage with health professionals primarily through networks available through the Medical School at ICL. For example, "Chemistry in the Clinic" an initiative at ICL, which fosters contact, interaction and exchange of ideas between clinicians and scientists, will provide an excellent opportunity to explore this aspect.

4. Public
We will also discuss our research findings and overall research area with the wider public by working closely with the press offices at ICL. For example, our recent work on DNA-binding pilins involved in Tfp-mediated natural transformation has been featured in press releases on ICL website (http://www.imperial.ac.uk/news) "Study finds how bacteria detect and ingest new DNA", and ICL iScience magazine (http://www.isciencemag.co.uk/) "A la carte DNA" and has attracted significant public interest.