Investigating the epigenetic regulation of pneumococcal virulence

In late winter the bacterium Streptococcus pneumoniae asymptomatically colonises the nasopharynx of up to seventy percent of children; it is also responsible for approximately 2.000.000 deaths worldwide due to complications of community acquired pneumonia. For over 80 years it has been recognized that when isolated from blood the bacterium forms opaque dome shaped colonies on solid laboratory medium whereas it forms flat transparent colonies when isolated from the nose. In general genetic regulatory events form the basis of the complex behaviour of any cell, both bacterial and eukaryotic; however no genetic differences could be identified between pneumococcal cells in the two distinct phase variable forms. Epigenetic regulatory mechanisms have in recent years been discovered to have a significant impact on bacterial cells. For the first time we have clear preliminary data that show that a phase variable epigenetic regulatory mechanism influences the capacity to colonise and cause invasive disease in this important human pathogen. The proposed project is aimed to elucidate the function of the phase variable restriction modification system responsible for this epigenetic regulation. The two main objectives instrumental to this aim are (i) to perform a molecular characterization of the mechanism by which the restriction modification system influences cell physiology and (ii) to describe the molecular mechanism by which the system recombines creating the epigenetically distinct subpopulations. Understanding and modelling the molecular nature of these processes is aimed to shed light on the intrinsic mechanisms by which a bacterium that is able to colonize asymptomatically in most children can rapidly change "behaviour" becoming one of the main human pathogens causing acute invasive disease. Such understanding is expected to be instrumental for designing improved intervention strategies both for prevention and treatment.

The important human pathogen Streptococcus pneumoniae has a clear in vitro phenotype which correlates with virulence, i.e. opaque colonies correlate to invasive infection and transparent colonies to carriage. We have clear preliminary data of an epigenetic phase variable regulatory system which has a major impact on virulence and upon the colony opacity phenotype. The mechanism of variation is based upon recombination within a restriction modification enzyme subunit gene which allows any pneumococcal population to separate into six coexisting genetically indistinguishable subpopulations.
The overall aim of the project is the determination of the mechanisms of epigenetic regulation of S. pneumoniae by the phase variable type I restriction modification system SpnIII. To achieve this aim we will investigate both (i) the molecular mechanisms by which SpnIII modulates gene expression and variations of pneumococcal phenotypes, and (ii) the function of the SpnIII HsdM and HsdR enzymes and the mechanism which allows the re-assortment of the six HsdS variants.
Technically we will use a series of mutant strains each expressing just one of the six possible HsdS hybrids, mine databases of target site conservation, exploit SMRT sequencing to define adenine methylation, use RNAseq to monitor gene expression and perform selective substitution of chromosomal methylation targets sites for validation. The functionality of SpnIII will be explored using gene-scan allele quantification, in vitro protein production, and modification and restriction of plasmids containing panels of synthetic SpnIII target sites. These two approaches should help to elucidate SpnIII epigenetic regulation and will allow mathematical modelling of the bistable regulatory circuit which is the basis of the pneumococcal colony opacity phenotype. The integration of biological data and modelling is expected to shed light on the significance of subpopulations in the virulence of this important human pathogen.

This research project addresses molecular mechanisms influencing fundamental aspects of the pathogenicity of the bacterium Streptococcus pneumoniae. One of the main beneficiaries is the wider public, since the project aims to unravel basic mechanisms involved in health and disease, but the project is also targeted for the benefit of biotech companies working in the fields of enzyme production or drug target development. Further beneficiaries include government and government agencies or regulators involved in setting up of epidemiological programs as our results may lead to important changes in or revision of pneumococcal typing or surveillance schemes. A further beneficiary is the wider scientific community as the project is a multidisciplinary project with many novel aspects and impact in a very wide range of scientific disciplines. An important beneficiary are, in addition to the early researcher within the project, young colleagues and the wider audience of students, academics and public addressed by our planned outreach activities. The interdisciplinary approach including other biologists and mathematicians, will clearly increase the skills of trainees enabling them to apply for jobs in a larger range of employment sectors.

S. pneumoniae is still, despite satisfactory vaccine coverage, one of the leading causes of mortality due to acute bacterial infection both in children and the elderly. This project has the potential to identify new drug targets or vaccine antigens. In contrast to most bacterial pathogens, there is for pneumococci a clearly defined in vitro phenotype (colony morphology), which matches virulence of the bacterium in humans. We plan in this work to characterise a system (potential drug target) that influences this phenotype, but more importantly we aim to identify the regulatory mechanisms (good potential as drug target) controlling this phenotype such as phase variation of SpnIII. In addition the restriction enzyme has shown in our preliminary characterisation peculiar specificities in substrate recognition. In accordance with the Business Development Office we will concentrate in the short term (1 year) on IPR protection and exploitation of the SpnIII enzyme and bacterial mutants and only later (2 to 3 years) on the molecular mechanism forming the basis of pneumococcal phase variation.

Regarding an impact on health and well-being, we are convinced that this investigation will provide a significant advancement in the understanding of pneumococcal physiology and its capacity to determine disease in the human host. Historically such advances have not always resulted in new treatment strategies, but novel drug discovery procedures and the interest in personalised and stratified medicine approaches provide a completely novel panorama for the discovery of relevant drug targets. Given the efforts put into targeted and stratified approaches in health technology, we strongly believe that this project has a high probability to impact very positively on the health of the UK citizens. Given the clear preliminary data, we think that the project may not only represent a novel approach but will lead to a breakthrough in work on this important human pathogen. The impact will not be immediate, but given the above considerations we envisage positive measurable effects on reduction of invasive pneumococcal disease and potentially also carriage in five to ten years,

A series of agencies are involved in drafting guidelines and in the planning and supervision of strategies for treatment, prevention and diagnostics of Streptococcal diseases. In this respect the important observation that the SpnIII system is not conserved in S. mitis, the non-pathogenic relative who forms with S. pneumoniae a single Operational Taxonomic Unit, is of interest. This difference might have impact on evolutionary concepts and diagnostics. In the latter case industries involved in bacterial diagnostics and typing might be affected.