Unraveling how N. meningitidis distinguish self from non-self DNA during DNA transformation

Natural transformation is the process by which bacteria, defined as genetically competent, take up naked DNA from the extracellular environment and incorporate it into their genomes by recombination. At least 60 bacterial species are naturally transformable, a number that is thought to be considerably underestimated. Natural transformation is an extremely valuable tool for research and was instrumental to one of the most important discoveries in biology (i.e. that the genetic material is DNA). Moreover, it is now clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity and a dominant force in bacterial evolution. This can have devastating consequences, e.g. by promoting spread of virulence or antibiotic resistance genes.

Type IV pili (Tfp), or related organelles, play a key role in DNA transformation. In addition, they are one of the most widespread colonization factors in bacteria. Therefore, Tfp have been and continue to be intensively studied as they might be primary targets for the development of new therapies against bacterial pathogens that place a heavy burden on human health and economy by infecting mankind, livestock and crops.

In current models, naked DNA needs to be bound by Tfp before it is taken up by the bacteria upon pilus retraction, but this remains to be formally demonstrated. The existence of a DNA receptor in Tfp is strengthened by the fact that in some species only self DNA is taken up because it contains a signature sequence motif known as the Uptake Sequence. In Neisseria, this DNA Uptake Sequence (DUS) motif is 10 bp long and is found in approx. 2,000 copies per genome. How competent bacteria bind DNA and are able to discriminate self from non-self DNA during uptake remains a mystery. The identification and analysis of a DUS receptor would represent a major breakthrough and would improve both our understanding of DNA transformation and Tfp biology in general.

This proposal originally derives from our recent characterization of N. meningitidis proteins involved in Tfp biology. Three of these proteins (ComP, PilV and PilX) are minor (low abundance) components of the Tfp. ComP plays a key role in competence. This prompted us to purify the ComP protein and test its DNA-binding activity, which led to a major finding: ComP has DNA-binding activity and shows a preference for the DUS (unpublished data). During this project, we plan to thoroughly characterize ComP's DNA-binding activity using biochemical and cellular assays, together with high resolution structural techniques.

Natural transformation is a widespread process by which bacteria, defined as genetically competent, take up naked DNA from the extracellular environment and incorporate it into their genomes. Natural transformation is an extremely valuable tool for research and was instrumental to one of the most important discoveries in biology (i.e. that the genetic material is DNA). Moreover, it is now clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity and a dominant force in bacterial evolution.

Type IV pili (Tfp) usually play a key role in DNA transformation. It is tought that DNA is bound by Tfp before it is taken up by the bacteria upon pilus retraction, but this remains to be formally demonstrated. In Neisseria species, the existence of a specific DNA receptor is strengthened by the fact that only self DNA is taken up because it contains a signature sequence motif known as the DNA Uptake Sequence (DUS), which is 10 bp long and found in ~ 2,000 copies/genome. However, how competent Neisseria bind DNA and are able to discriminate self from non-self DNA remains a mystery. The identification and analysis of a DUS receptor would represent a major breakthrough and would improve both our understanding of DNA transformation and Tfp biology in general.

Our recently published systematic characterization of N. meningitidis genes involved in Tfp biology identified three minor (low abundance) pilins, one of which (ComP) plays a key role in competence. This prompted us to purify the ComP protein and test its DNA-binding activity, which led to a major finding: ComP has DNA-binding activity and shows a preference for the DUS (unpublished data). In this research project, we will (i) characterize thoroughly ComP's DUS-binding activity, (ii) determine a high resolution structure of ComP and the ComP-DUS complex and (iii) perform a detailed structure/function analysis of ComP's DUS-binding activity.

Although proposing basic "blue-sky" research, this project will have a more general impact because it will lead to a better understanding of arguably one the most widespread virulence factors in bacteria. i.e. Tfp. Tfp might be present in 150 different species spanning most bacterial phyla and are the only pili present in both Gram-negative and Gram-positive bacteria, including many pathogens. As such, Tfp are primary targets for the development of new therapies against bacterial pathogens that place a heavy burden on human health and economy by infecting mankind, livestock and crops. Our work might thus benefit large Pharma (Pil proteins might be targets for the design of drugs interfering with the important functions mediated by Tfp), smaller Biotech companies (some of the NMR methodology/expertise developed during this project is likely to spill over into the BBSRC-funded CASE collaboration SM has with Arrow therapeutics) or not-for-profit organizations that are involved in developing new vaccines against pathogenic bacteria (minor pilins such as ComP might have vaccine potential against N. meningitidis as shown recently by VP's group).

We will, if necessary, ensure that intellectual property opportunities are maintained through liaison with technology transfer expertise teams at BBSRC and Imperial College (IC INNOVATIONS Ltd).