Investigating the epigenetic regulation of pneumococcal virulence

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description Academic Collaboration Agreement
Amount £140,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 12/2017 
End 11/2019
 
Description ESCMID study group grant
Amount € 30,000 (EUR)
Organisation European Society of Clinical Microbiology and Infectious Diseases (ESCMID) 
Sector Learned Society
Country European Union (EU)
Start 03/2016 
End 12/2017
 
Description Fee for Service Contract
Amount £53,000 (GBP)
Organisation F. Hoffmann-La Roche AG 
Sector Private
Country Global
Start 01/2018 
End 04/2018
 
Description KTN CASE BBSRC
Amount £98,000 (GBP)
Funding ID BB/P504737/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Wellcome Trust equipment fund
Amount £290,555 (GBP)
Funding ID 208393/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 12/2021
 
Title Mathematical model to calculate recombination frequencies and predict experimental outcome 
Description We have constructed a mathematical model to be able to predict the results of recombination in a bacterial population after an arbitrary number of divisions and an arbitrary initial genetic composition. The considered model is a Markov chain model using a transition matrix which accounts for the recombination frequency. The elements of the transition matrix were expressed in terms of the probabilities of DNA inversions on three distinct inverted repeats by repeated binary space partition. The model is bale to compute recombination frequencies and to predict recombination and allele frequencies at experimental outcome. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact to predict experimental outcome of phase variable populations with respect to the phase variable genes a tool is needed to calculate and predict experimental outcome given a standard set of parameters. Only against such data it is possible to evaluate then if a given experimental oucome is as expected or different from expected. 
 
Title Organotypic slice culture for evaluation of bacterial growth in macrophages 
Description The work on bacteria phase variation led us to study bottlenecks during infection and to the identification of within-macrophage replication. In our recent paper, we have described that just minutes after in vivo challenge of mice, 'typically-extracellular' bacteria efficiently replicate in tissue macrophages (Ercoli Nature Microbiology 2018). Subsequently, we have realised that once bacteria have been delivered in vivo to splenic tissue macrophages, the organs can be collected, sliced and cultured ex vivo in the laboratory. In these tissue slices - which conserve the 3D microarchitecture - we observe that bacteria continue to replicate within the macrophages (unpublished). The availability from a single infected organ to prepare approximately forty slices in which bacteria are rapidly replicating within cells, and which can be conveniently incubated in flat bottomed micro-titre plates, prompted us to attempt to exploit this tool for within-tissue susceptibility testing. Our unpublished data show that a 6-hour incubation of the tissue slices in the presence of serial dilutions of antibiotic allows for detection of bacteriostatic and bactericidal activity of the drugs in question. The format of the method is comparable to a classical microbroth dilution assay, with the exception that one infected organ slice (obtained 30 minutes after in vivo challenge) is incubated in each microtitre well. Antimicrobial drugs are then added as in standard susceptibility testing and minimal inhibitory concentrations (MIC) and minimal bactericidal/fungicidal concentrations (MBC/MFC) are detected by bacterial colony counting. After six hours of incubation bacterial enumeration allows determination of growth, inhibition and bactericidal activity of a drug. Our pilot data show that pharmacokinetic drug penetration ratios correlate well with the experimental intra-extra-cellular drug efficacy ratios determined by our method on a model strain. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact An outline grant on the use of organotypic slice as an antimicrobial drug activity testing method during the phases of hit characterisation and hit to lead development. This project has been invited recently by NC3R for full submission. We have also proposed organotypic slice cultures to GSK Vaccines as novel correlate of protection to be used during screening for vaccine antigens. We are now applying jointly with GSK for funding to proof the effective utility of this methodology. 
 
Title Quantifying Streptococcus pneumoniae for the six-alleles system SpnD39III 
Description In any given wild type population of Streptococcus pneumoniae the Type I Restriction Methylation System (SpnD39III) can be found to exist in six different allelic forms. This work describes a protocol for allele quantification for these SpnD39III forms which is composed of three main steps: an initial common PCR, a differentiating restriction digestion, and then a quantitative GeneScan analysis. The PCR uses primers, one of which is fluorescently labelled, which are sited in flanking regions that are common for the six allelic variants; this is followed by a digestion with two commercial restriction enzymes that restrict the different DNA sequences of each of the forms giving rise to six different-sized labelled fragments. The digested samples are then analysed on a GeneScan platform giving a spectrum of peaks whose areas can be compared, thereby allowing the relative quantification of the six SpnD39III forms. Using this protocol we are able to determine the SpnD39III allele composition of any pneumococcal sample in less than 3 days. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact the idea is to publish the methodology and then to licence out the know-how in performing the test 
 
Title Tissue slice cultures for improved intracellular antimicrobial drug-activity testing. 
Description Our work on phase variation in the host has made us discover within-macrophage replication of typically extracellular bacteria. This led to the discovery that shortly after challenge spleens can be harvested and tissue slice cultures set up. This has a drammatic impact on 3Rs as it reduces anmila numbers as 40 slices can be generated form each sample thus reducing numbers by a factor of 40 and in addition it significantly refines systemic disease models as samples can be taken 30 minutes after challenge, when animals disply no signs of disease. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact In the development of antimicrobial drugs, in vivo testing of efficacy represents a it is critical to determine their penetration to, and activity within host cells, as these are key determinants of clinical efficacy. In this project we propose an entirely new method for the intratissue and intracellular activity testing of antimicrobial drugs, that fulfils the principles of REDUCTION and REFINEMENT. Our method uses ex vivo spleen slice cultures, an organ which is both critical to host defence against infection, but which is also a reservoir for bacteria which can result in an infectious relapse. In drug development programmes, the new method will decrease the number of compounds progressed to in vivo screening, by introduction of an ex vivo screening that will eliminate ineffective candidate compounds, thereby REDUCING whole animal testing. As approximately forty slices can be obtained from a single murine spleen, this method further REDUCES the number of experimental animals in preclinical antibiotic research by a forty-fold REDUCTION in animals required for ex vivo testing. The new method represents a significant REFINEMENT, that decreases animal suffering. To enable drug efficacy to be demonstrated in current in vivo models in invasive infection and sepsis infection progresses to a stage where the signs of disease reach the 'moderate' to 'severe' stages. Because our methodology relies on tissue sampling only 30 minutes after infection, it refines the current gold standard infection models significantly by avoiding any signs of disease. Thus, the new method is classified as 'mild'. Our method is aimed to refine anti-infective development practices during the phases of hit characterisation and hit to lead development. Once validated, this 3Rs compliant intracellular testing method will provide a welcome tool to screen novel molecules because it reduces animal numbers and costs for pharmaceutical companies, contract research organisations and academic groups involved in anti-infective screening. To illustrate the potential for REDUCTION, from our own contract research experiences, we predict to test 50 compounds at five dilutions against four clinically relevant reference bacteria, in triplicate, would require of 60 mice for our new method instead of 2,400 mice by current methods. Once validated, the methodology will be easily transferred to other laboratories as the experimental methodology is straight forward. When expanding to the UK, or on a world-wide basis, this is an enormous reduction in experimental animals. Following the recent worldwide 'state of emergency' on the dramatic potential impact of antimicrobial drug resistance on worldwide health, the UK government, as well as other national and international organisations, has decided to increase public funding for research into new antimicrobial drugs. Our method will provide a strategic tool to optimise the within tissue and within cell activity of compounds early in the drug development process. This utility will be a strong selling point for CROs and academic groups involved in compound screening. 
 
Description Bacteriophages as indicators of phase variable restriction systems 
Organisation University of Leicester
Department Department of Infection, Immunity and Inflammation
Country United Kingdom 
Sector Academic/University 
PI Contribution we isolated pneumococcal bacteriophages and used them to test for the activity and variant of the methylation system by testing restriction
Collaborator Contribution we gaind insight into phage biology and methodology
Impact a manuscript has been submitted: Furi L, LA Crawford, G Rangel-Pineros, A Sousa Manso, M Galati, M De Ste Croix, R Haigh, K Engelsen Fjelland, G Duncan Gilfillan, W Ting Chan, M Espinosa, M Clokie, MR Oggioni. Methylation warfare; interaction of streptococcal bacteriophages with their host. Submitted.
Start Year 2016
 
Description Defining the molecular steps in the interaction of pathogenic bacteria with alveolar macrophages a key step in pathogenesis and disease. 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution BBSRC-GSK CASE CSV grant BB/S507052/1. The industrial partner is the preclinical research centre of GSK Vaccines in Siena, Italy. Our contribution relates to the elucidation of the role of phase variation of the bacteria in the host and within-macrophage replication.
Collaborator Contribution know how on vaccines and correlates of protection
Impact work is ongoing. The first abstracts have been submitted to conferences.
Start Year 2018
 
Description Large scale genomic analysis shows no evidence for pathogen evolution during late stage bacterial meningitis in humans 
Organisation Columbia University
Department Department of Neurology
Country United States 
Sector Academic/University 
PI Contribution development of nested real time PCR protocol for quantification of phase variable genes. This enabled to test prevalence of phase variable genes both directly in clinical samples and in bacteria grown from those samples. This data showed prefect correlation of allele prevalence between clinical samples and bacterial stock, which were used for genome sequencing and this in turn allowed to deduce allele prevalences from large set of bacterial genome sequences in silico.
Collaborator Contribution The Sanger Centre sequences hundreds of bacteria and performed bioinformatic analysis. The AMC collected all bacterial strains and had clinical data to which to correlate genomic data.
Impact The manuscript Lees et al., Large scale genomic analysis shows no evidence for pathogen evolution during bacterial meningitis in humans. has been submitted to PLoS Genetics.
Start Year 2015
 
Description Large scale genomic analysis shows no evidence for pathogen evolution during late stage bacterial meningitis in humans 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution development of nested real time PCR protocol for quantification of phase variable genes. This enabled to test prevalence of phase variable genes both directly in clinical samples and in bacteria grown from those samples. This data showed prefect correlation of allele prevalence between clinical samples and bacterial stock, which were used for genome sequencing and this in turn allowed to deduce allele prevalences from large set of bacterial genome sequences in silico.
Collaborator Contribution The Sanger Centre sequences hundreds of bacteria and performed bioinformatic analysis. The AMC collected all bacterial strains and had clinical data to which to correlate genomic data.
Impact The manuscript Lees et al., Large scale genomic analysis shows no evidence for pathogen evolution during bacterial meningitis in humans. has been submitted to PLoS Genetics.
Start Year 2015
 
Description Phase variation during human carriage of Streptococcus pneumoniae 
Organisation Liverpool School of Tropical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution the Experimental Human Pneumococcal Carriage (EHPC) collaboration is a 5 year MRC program grant initiative. In order to explore phase variation during carriage we were given access to serial samples of 42 individuals. Using these samples we were able to qualify active alleles of our phase variable methylation system in over 100 human samples. This allowed to mathematically model phase variation in humans.
Collaborator Contribution the Experimental Human Pneumococcal Carriage (EHPC) collaboration is a 5 year MRC program grant initiative. In order to explore phase variation during carriage we were given access to serial samples of 42 individuals. Using these samples we were able to qualify active alleles of our phase variable methylation system in over 100 human samples. This allowed to mathematically model phase variation in humans.
Impact a manuscript is being finalised
Start Year 2017
 
Description Susceptibility to complement 
Organisation F. Hoffmann-La Roche AG
Department Pharma Research and Early Development
Country Switzerland 
Sector Private 
PI Contribution testing of bactericidal activity of human sera. Our expertise came form our studies on infection models.
Collaborator Contribution testing of reagents
Impact confidential data
Start Year 2018
 
Description Testing new correlates of protection for an Escherichia coli vaccine 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution our work on phase variation in the host made us discover within-macrophage replication. We are no proposing this as new correlates of protection for the screening of bacterial vaccine antigens.
Collaborator Contribution Provision of antigens and sera and know how on correlates of protection
Impact a joint grant application is planned to be submitted in May 2019 to the MRC funded network Bactivac
Start Year 2019
 
Description Tracking intracellular pneumococci in humans 
Organisation Queen Elizabeth Central Hospital, Malawi
Country Malawi 
Sector Hospitals 
PI Contribution We will perform confocal and electron microscopy and bacterial typing.
Collaborator Contribution The Malawi-Liverpool-Wellcome Trust Clinical Research Institute placed at the main hospital in Blantyre, Malawi will collect human bronco-alveolar lavages
Impact Work includes clinical bronco alveolar sample taking.
Start Year 2018
 
Title Short course treatment for low severity Community Acquired Pneumonia 
Description The work on bacterial phase variation during early infection has brought to the unexpected discovery of bacteria replicating within macrophages prior to invasive bacterial disease and sepsis. By keeping testing this in mouse models, we could show that the choice of antibiotic during the early stages of infection is crucial for prevention of late onset bacteraemia. Together with a group of clinical colleagues, we are now in the process of applying to the NIHR HTA scheme for a Feasibility Trial to test short-course antibiotic treatment for low-severity community acquired pneumonia. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2016
Development Status Actively seeking support
Impact this will depend on the outcome of the clinical trial planned 
 
Title Tissue Models for Invasive Disease (TIMID) 
Description Our work on phase variation made us discover within-macrophage replication of bacteria and we have published this for mice and pigs. This led us to apply for a trial to explore fate of bacteria in the human spleen during the early stages of infection. This trial has been granted and work commenced in summer 2018. Study Title: Tissue Models for Invasive Disease (TIMID) Chief Investigator: Marco R Oggioni Site: University Hospitals of Leicester NHS Trust REC reference: 18/EM/0057 UoL Ref: UOL 0640 IRAS project ID: 219992 End date: 28/02/2021 
Type Therapeutic Intervention - Vaccines
Current Stage Of Development Initial development
Year Development Stage Completed 2018
Development Status Under active development/distribution
Impact Work on the phase variation of S. pneumoniae during infection made us discover the intracellular phase of these bacteria. This led to the writing of the TIMID clinical trial. With this unique set up we plan to apply for new funding from the MRC in May 2019. 
 
Title Epigenetics Mobile Application: Germ Generator 
Description Epigenetics Mobile Application: Germ Generator. The purpose of the mobile game is to educate and engage the public and young people on the role of epigenetics in Streptococcus pneumoniae virulence in the human body. The game will function as an educational but engaging game and will be utilised in public engagement events and on-going promotion of University of Leicester research. A V2 development has been made and the app prototype is downloadable and has been used at DYNAMIC DNA, the ooutreach for schools of the University of Leicester Genetics initiative. We are sourcing further funding to complete the design so that the app can be fully developed and be freely downloadable for the app store. 
Type Of Technology Webtool/Application 
Year Produced 2018 
Impact The initial work to develop and test a prototype app has been funded using BBSRC grant BB/N002903/1 and is based on our work in MR/M003078/1 and BB/P504737/1.After a first proptotype was designed and tested, the aim of prototype v2 is to investigate effectiveness of the core game mechanic (spawning and functions) and to test effectiveness of Design Lab user interface. In addition, prototype v2 also aims to assess intuitiveness of game. The game is aimed to be be published on Google Play and Apple application stores, making it freely available for researchers, teachers and other outreach educators to use as a tool to teach microbial genetics. The target audience of the app is KS3-4 (11-16 years old) pupils however it will be suitable for use by the public in general and can be used as a learning tools independently of UoL researchers. We hope the full version of the app will encourage students to learn that specific microbial phenotypes interact with the immune system in different ways. This will allow them to begin thinking beyond the school curriculum which predominantly confines bacteria to the study of "health, disease and the development of medicines". 
 
Description Epigenetic training in schools 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Megan De Ste Croix of my team is teaching with The Brilliant Club in 2016 to 2018 to a variety of age groups (Year7-9). Courses focus on microbiology, infection and epigenetics. All courses are designed to be beyond the standard curriculum for the age group taught. The specific aim of the brilliant club is to raise aspirations to attend top universities.
Year(s) Of Engagement Activity 2016,2018
 
Description Modelling Biological Evolution 2017: Developing Novel Approaches (April 2017 Leicester) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The project is a collaboration with the Department of Genetics and the Department of Mathematics. The CoI Andrew Morozow is organiser in Leicester of the International conference on Modelling Biological Evolution 2017 (University of Leicester (UK), April 4th -7th, 2017). This is an impartant aspect in our attmpt to strengthen an interdisciplinary dialogue. The conference is supported by this grant.
Year(s) Of Engagement Activity 2017