Understanding DNA-binding by type IV pilins: key event during transformation in naturally competent bacteria

Natural transformation is the biological process during which bacteria capture free DNA from their surroundings and incorporate it into their genomes to become genetically altered by acquiring new genes. This process has important implications for human health because it promotes horizontal transfer of genes between bacteria, which can have devastating medical consequences when it leads to the emergence of bacterial pathogens with increased virulence and/or resistance to antibiotics.

The first step during natural transformation, DNA uptake, is mediated by surface filaments named Tff composed of subunits called type IV pilins. These filaments are extremely important because they (i) are almost universal in prokaryotes, and (ii) have been associated with a fascinating array of very different properties, many of which are key for the virulence of bacteria causing important human diseases such as cholera, meningitis, gonorrhoea, diarrhea etc. Studying Tff biology is of great medical interest in this era of rising antibiotic resistance since this could ultimately lead to the development of new antivirulence drugs interfering with Tff assembly or Tff-mediated functions.

Until recently, the earliest step in transformation, how Tff interact with DNA was poorly understood because no Tff-associated DNA receptor had ever been identified. Our recent work provided a breakthrough by showing that type IV pilins related to meningococcal ComP display intrinsic DNA-binding ability and bind DNA in a novel fashion. This paves the way to an atomic level understanding of how type IV pilins bind DNA during natural transformation, which is the overarching objective of this research proposal.

Using a combination of genetic, biochemical, and structural approaches for which we have extensive experience, we will address the following three aims.

Aim 1: determine at an atomic level how ComP pilins bind DNA and how different orthologs are able to distinguish between closely related DNA motifs.

Aim 2: characterise the recently identified Pasteurellaceae DNA-binding pilin, unrelated to ComP, to determine how it binds DNA.

Aim 3: identify the DNA-binding pilin in competence (pseudo)pili, the most widespread Tff involved in natural transformation.

Natural transformation is the biological process widespread in bacteria during which some species capture free DNA from their surroundings and incorporate it into their genomes. This process can have devastating medical consequences by leading to the emergence of bacterial pathogens with increased virulence and/or resistance to antibiotics.

The first step during natural transformation, DNA uptake, is mediated by surface nanomachines composed of type IV pilins, which are known as type IV filaments (Tff). Tff are almost universal in prokaryotes and have been associated with a vast array of properties, many of which are key for the virulence of bacteria causing important human diseases.

Our recent work has explained how Tff interact with DNA. We have identified the first type IV pilin with intrinsic DNA-binding ability, ComP from N. meningitidis. We found that ComP specifically binds the DUS motif, which in meningococci promotes selective uptake of homotypic DNA. We found that this important mechanism of protection against indiscriminate transformation by foreign DNA is shared by most species from the Neisseriaceae family that harbour ComP homologs recognizing different DUS. The high-resolution structures we determined for two ComP orthologs identified a novel mode of interaction with DNA.

We will address two important questions during this project. How can different ComP orthologs differentiate between closely related DUS motifs? How do other DNA-binding pilins, in competent bacteria where there are no ComP homologs, interact with DNA. This is expected to generate a holistic view on DNA-binding by type IV pilins, which will have important and broad implications for nanomachines that are virtually ubiquitous in prokaryotes. This could ultimately lead to the development of new antivirulence drugs interfering with Tff assembly or Tff-mediated functions, which are needed in this era of rising antibiotic resistance.

The outcomes of this fundamental biology research project will be the elucidation of the fascinating process by which type IV pilins bind DNA-binding during the earliest and key event in transformation in naturally competent bacteria. Importantly, many of these competent species, including the human pathogens N. meningitidis, N. gonorrhoeae and H. influenzae are of medical relevance. In addition, this project will lead to a better understanding of the biology of Tff nanomachines, which are virtually ubiquitous in prokaryotes (Bacteria and Archaea). 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. The applicants VP and SM will take joint responsibility for maximizing impact. This is detailed in the Pathways to impact document.

1. Academia
We will ensure that our research is disseminated widely by Open Access publication in high-impact journals and presentation at international research conferences. All structural data will be freely available online through standard repositories.

2. Pharma, biotech companies, public sector health care professionals
The proposed research is not expected to generate commercially or medically exploitable results within the time frame of this project. However, should such opportunities emerge in the future, we will after devising an appropriate IP protection strategy in liaison with MRC's and ICL technology transfer expertise: (i) target large Pharma and smaller biotech companies with the help of business and translational offices at ICL, (ii) participate in the national network for Protein-Protein Interactions, (iii) contribute to industrial open-days at ICL, and (iv) engage with health professionals through networks available at ICL, such as "Chemistry in the Clinic".

3. Public engagement
We will continue to discuss our research findings and overall research area with the wider public. SM has made school-oriented presentations to explain how structural biology assists in drug development and scientific discovery. He will endeavour to continue this approach. The applicants will contribute to Schools Open Days on the ICL Campus for both pupils and teachers. They will also work closely with the press offices at ICL to disseminate via the popular science press and other media formats, any specific discoveries of general interest. Importantly, our work on ComP has attracted significant public interest and 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".

4. Skills, training and knowledge economy
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. They will be trained in key techniques and good laboratory practice, encouraging innovative approaches to research. The PDRA will also benefit from the highly active staff development programme at ICL, which includes courses on presenting science to a lay audience, developing an independent research career, grant writing, exploiting translational aspects of research etc.

5. Collaboration
The project will strengthen an already successful collaborative relationship between the VP and SM groups, and will benefit both groups. We will continue to have regular research meetings every two weeks. The current developments in both groups are likely to fuel future collaborations.