Phase variable epigenetic control in firmicutes

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

All the cells of a species contain the same DNA; what distinguishes them is the way in which that DNA is activated ('transcribed'). One method of regulating DNA transcription is through direct chemical modification of the DNA itself, most commonly through a process of methylation. This is termed 'epigenetic' regulation. There has been extensive study of epigenetic regulation in humans and other complex life forms, but comparatively little in bacteria. Bacterial genomes are often extensively methylated, as a consequence of 'restriction modification' (RM) systems, which modify the cell's DNA at particular sites to allow it to be distinguished from the DNA of infecting viruses. The many sites of methylation across the genome have the potential to substantially affect the way in which genes are regulated. However, as most RM systems are stable and therefore cannot serve as a regulatory mechanism. This is not the case for two sets of genes we have recently independently characterised in the bacterium Streptococcus pneumoniae, the pneumococcus. The pneumococcus is a commensal bacterium, typically carried by between a quarter and a third of young children asymptomatically, that is a major cause of diseases including middle ear infections, pneumonia and meningitis. The sets of genes we found are RM systems that vary over the course of hours or days through a specific set of DNA rearrangements. This results in the patterns of methylation caused by the RM systems also changing over short timescales. Experimental data found that different forms of the inverting RM system caused different patterns of methylation, each of which was associated with a distinct pattern of gene expression. This epigenetic regulation of bacterial genes was found to change the virulence of the bacterium, with some patterns of methylation making the pneumococcus more likely to cause disease. This could be an important factor in the transition from the pneumococcus being a harmless commensal, to becoming a dangerous pathogen. This project is designed to test this hypothesis through studying whether the second variable RM system in the pneumococcus affects the same processes, or has a different effect on cell physiology, and whether such systems regulate the virulence of other pathogenic bacteria. Searching of the thousands of publically available bacterial DNA sequences has allowed us to identify hundreds of species that harbour similar systems. These include bacterial species that are very common in the human gut, some that are present in probiotic drinks and others involved in the production of cheese. Perhaps most importantly, they are also present in many pathogenic bacteria. This project is designed to investigate whether these variable RM systems might also regulate virulence in three bacterial species that each represent major threats to public health. The first is Streptococcus suis, a species normally associated with pigs that is emerging as a major pathogen capable of causing serious infections, such as meningitis. The second is Listeria monocytogenes, a foodborne bacterium that causes potentially fatal infections. The third is Enterococcus faecalis, a major cause of highly antibiotic-resistant infections, particularly in a hospital setting. In the three species, the variable RM loci are present with lineages that are associated with causing high levels of disease in humans, and absent from those that are asymptomatically found in animals or humans. The overall aim of the project is to work out how these systems may play a role in regulating genes involved in the bacteria's virulence, as well as how they evolved and how diverse they are. Such information will allow us to understand why these unusual genes are distributed, and why bacteria progress from being harmlessly carried to causing disease. This would better inform our strategies as to how to prevent this transition, and thereby tame these common, but potentially dangerous, bacteria.

Technical Summary

Two distinct phase variable type I restriction modification systems, able to change their specificity through shuffling of sequences facilitated by a recombinase, have been described by us in the pathogen Streptococcus pneumoniae in two independent papers (Manso et al., Nat Commun. 2014, 5:5055; Croucher et al., Nat Commun. 2014. 5:5471). One of these was found to be responsible for phase variable epigenetic modification of the microbial chromosome with impact on gene expression and virulence. Both loci are absent from the related non-pathogenic species, S. mitis. We now have mined available genome information and detected four families of recombinases associated with Type I RM loci across many species, all of which are predicted to be able to vary the patterns of methylation over short timescales. Although phase variable RM loci can be found in many phyla, they are particularly highly represented among Firmicutes. In particular we have collected detailed information on likely phase variable systems in Listeria monocytogenes, Enterococcus faecalis and Streptococcus suis. In all three cases the lineage or clonal complex most frequently found to cause human invasive infection is associated with the presence of a phase variable Type I RM locus. The aim of the project is to analyse the overall evolution of these systems, and then isolate clones expressing different allelic variants in the four species of interest on which to perform quantification of phase variation, methylome analysis, gene expression profiling associated to metabolome assays and virulence tests in experimental infection models. These tests are designed understand the biological mechanism underpinning the epigenetic regulation of important phenotypes. Demonstration of phase variable epigenetic control in these four species is predicted to allow us to propose phase variable epigenetic control by type I RM systems as global and novel paradigm of bacterial evolution, physiology and virulence.

Planned Impact

This research project is designed to test if the novel epigenetic control mechanism discovered in Streptococcus pneumoniae is an unicum of this species, as published independently by us in two high impact papers (Manso et al., Nat Commun. 2014, 5:5055; Croucher et al., Nat Commun. 2014. 5:5471), or if holds true to be a paradigm of epigenetic control for a wide number of bacteria. We have clear preliminary data that show that these phase variable type RM systems are broadly distributed, and likely to be active, in many bacterial phyla.

One of the main beneficiaries is the wider public, since the project aims to unravel basic mechanisms involved in bacterial population dynamics and more importantly showing that phase variable epigenetic control is widespread also in bacteria. In the specific case we address four bacterial species of substantial interest to the BBSRC, industry and health care professionals.

Interest in food safety is a primary goal for all players in the long chain and the species Listeria monocytogenes has been selected as an important food borne pathogen. Data on phase variable RM systems in Listeria are hoped to yield novel epidemiological markers, markers associated to disease severity but also shed light on the virulence of this species. The concept linking presence of phase variable RM systems to highly pathogenic lineages will be tested in also S. suis, a zoonotic pathogen and first cause of human meningitis in South-east Asia. In this species the clear association of the phase variable type I RM system to serotype 2 strains, could well turn out to be one of the important virulence determinant.

The UK has recently launched the 5 Year Antimicrobial Resistance Strategy 2013 to 2018. One of the important aspects in this strategy is to limit drug resistance to develop, but more importantly to spread. Of particular concern is the fact that many more antibiotics are used for industrial animal husbandry than directly for humans, which in turn has driven an alarming rise in antimicrobial drug resistance in bacteria carried by animals. Hence the final model species included in this project is Enterococcus faecalis, among the main candidates responsible for transfer of antimicrobial drug resistance from animals to humans.

The widespread nature of these gene modules will allow transferring information more widely, as these gene clusters are found, in the two main phyla found within the human intestinal tract, in many probiotic and dairy species. Given the clear preliminary experimental data and the broad range of bacteria being investigated, this scientifically novel project is highly relevant to the study of bacteria globally. The industrial or health related impact may not be immediate, but given the above considerations we envisage potentially positive measurable effects in terms of public health and the field of food safety and production within few years after the project's completion.

The interdisciplinary and multi-centre nature of this project means it will be effective in training researchers in a wide range of skills. They will learn the latest molecular microbiology techniques, how to analyse high-throughput sequencing data, mine available genome sequences, and exploit newly-available sequencing technologies. Collaborations with the broader community of researchers, as outlined in our proposal, will provide for interactions with clinicians and public health experts. Other potential beneficiaries include the wider audience of students, academics and public addressed by our planned outreach activities.

Discovering novel regulatory mechanisms, which may have the potential to be valid for many species holds promise to identify potential drug targets and inhibitors. This will not be addressed in depth within the project, but throughout the research we expect to get critical scientific information that may need protection and consideration for commercialisation.

Publications

10 25 50

 
Description Summary for a lay audience: The main finding of our work on a phase-variable DNA methylation systems in bacteria is that epigenetic modification near the start of a gene increases the expression of that gene. An important difference to published work, is that our data show that methylation of DNA, not only in the specific sequences denominated "promoter" or "regulator binding site", can determine an increase of expression of the downstream genes. We have comparable data for three different genes in the genome of Streptococcus pneumoniae and have controlled the observed variation in gene expression by generating recombinant bacteria in two unrelated bacterial strains. These data represent a novelty as they are the first demonstration of changes in gene expression driven by adenine methylation by restriction modification systems and a great step forward in the analysis of epigenetic gene regulation in bacteria. These data are presently being collated and prepared for submission for publication. In previous publications reporting the work of this grant (De Ste Croix FEMS Microbiol Rev 2017), we have already described the recombination mechanism of these phase variable systems (Kwun NAR 2018; De Ste Croix et al., J Bact 2019), their variation during colonisation of humans (De Ste Croix et al., SciRep 2020), and their impact on bacteriophages and transformation (Kwun et al., Genes 2019; Furi et al., J Bact 2019).

Specific workpackages addressed:
Phase variable methylation impacts gene expression in Listeria monocytogenes.
The aim of this work is to determine what impact alternative DNA methylation patterns have on gene expression in L. monocytogenes. We found that the phase variable RM system recombined only at temperatures below 30 C. This enabled to isolate stable clones expressing only one of the four methylation specificities form three CC4 strains N12-0394, N12-0974 and 2252048. In all three clinical isolates the B-variant displayed a lysis related phenotype in stationary phase. Through PacBio sequencing we have determined the methylation patterns of four alternate hsdS genes in a L. monocytogenes CC4, CC5 and CC8 strains with a PV Type I RM system. Subsequent RNAseq analysis of CC4 strains expressing a single hsdS gene identified differentially expressed genes with methylation sites in their promoter regions. Using a luciferase reporter plasmid we have created wildtype and modified promoters to investigate the impact of promoter methylation on gene expression. In the CC4 strain N12-0320, methylation sites within the lytC promoters appear to alter the expression of downstream genes. While not ubiquitous like the pneumococcal system, we have shown that hsdS shuffling in L. monocytogenes is temperature dependent. Importantly, environmental conditions can alter the proportions of hsdS variants in a population resulting in differential gene expression through PV methylation. A Manuscript is being prepared.

RM systems drive sub-lineage specific evolution in Listeria monocytogenes.
We have sequenced a panel of 160 Swiss L. monocytogenes isolates from food and human origin. We detected five phase variable type I RM systems which were present in five of the nine epidemic clones responsible for outbreaks in central Europe. In contrast non-phase-variable type I RM were not associated with outbreaks. This statistically significant difference could indicate a fitness gain of isolates carrying phase variable type I RM system. In addition, a clear sub-lineage specific distribution of all RM systems was noted indicating that acquisition of RM systems is at the origin of sub-lineage evolution. Pairwise analysis of SNP and cgMLST data of 700 strains indicated sub-lineage differences in the cut off for definition of outbreaks and differences in mutation rate and recombination between lineages. This difference in recombination intriguingly shows a barrier to genetic exchange between lineages I and II, while exchange to and from lineage III is more frequent. A Manuscript is ready for submission.

Phase variable methylation impacts gene expression in Enterococcus faecalis.
A genomic survey of phase variable type I RM systems in E. faecalis has shown two different families, with six and 9 subfamilies of systems which harbour different alleles of specificity genes. Two of the main drug resistant hospital acquired clonal complexes have a conserved phase variable type I RM system in all isolates. As in Listeria, the enterococcal phase variable RM system in CC8 was found to recombine only below 30 degrees which again enabled to isolate stable clones expressing a single methylation specificity. Pacbio sequencing and RNAseq allowed to determine the target sites of the phase variable system and document differences in gene expression between the four variants. qPCR confirmation is ongoing. A Manuscript is being prepared.

Characterisation of the Streptococcus pneumoniae SpnIV phase-variable Type I RM system.
The SpnIV phase-variable Type I RM system of Streptococcus pneumoniae, encoded by the translocating variable restriction (tvr) locus, has been extensively characterised in terms of its target motifs, mechanism and regulation of phase variation, as well as its effects on exchange of sequence through transformation. This work has been published as Kwun et al (2018) Nuc. Acids Res. 46(21):11438-11453. The specificity-determining hsdS genes were found to be shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus makes important contributions to the variation in genome methylation both within and between strains, and at the population level will function to limit the genomic plasticity of S. pneumoniae strains.

Methylation dependent gene expression in Streptococcus pneumoniae.
A panel of five independent isolates was generated which contained 80 to 90% of cells expressing one of the phase variable specificity genes. These panels of five strains were generated for hsdS variant A, B and C and gene expression was analysed by RNAseq. The promoter regions of three differentially expressed genes which had a methylation site in or near the promoter were synthesised and cloned in front of a luciferase reporter. Promoters with a SNP abolishing methylation or conferring altered methylation specificity were also synthetized and cloned. Data three promoter constructs show that methylation of the promoter, at the +1 of within a 5'UTR increases luciferase reported production. When changing the methylation target site specificity, the reporter genes were now expressed more in bacterial strains with a that methylation specificity and less in the original wt strain. We have transferred constructs into an unrelated S. pneumoniae serotype 19F starin and observed the same variation of gene expression depending on the methylation specificity of the recombinant hots. These data represent a novelty as they are the first demonstration of changes in gene expression driven by adenine methylation by restriction modification systems and a great step forward in the analysis of epigenetic gene regulation in bacteria.
Otherwise-isogenic S. pneumoniae mutants with different tvr loci have been found to exhibit phenotypic differences in growth patterns and transformability in two backgrounds. Removal of the tvr locus eliminated the phenotypic difference in both cases, strongly suggesting the differing arrangements of the SpnIV restriction-modification system causes the phenotypic change. In one of these backgrounds, we have identified an apparent interaction between a mobile genetic element and the SpnIV locus that correlates with the change in phenotype. Characterisation of the mechanism linking the different arrangement with the phenotype is ongoing. Samples for RNA sequencing will soon be sent to the Wellcome Sanger Institute.

A Review was published describing Phase-variable methylation and epigenetic regulation by type I restriction-modification systems. (De Ste Croix et al., FEMS Microbiol Rev. 2017)
Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that, through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria.

Bioinformatic analysis of phase-variable restriction-modification systems:
Methods have been developed to enable scanning of the entire database of bacterial draft genome sequences for phase-variable Type I restriction-modification systems. This has enabled us to identify five distinct families of these systems, characterise the ancestral sequences from which they evolved, and identify their distribution across bacterial species. This found representatives of these restriction-modification systems across multiple Phyla of bacteria. We have also constructed networks to illustrate the patterns of similarity between the constituent proteins of the systems. This demonstrates they have moved between diverse species relatively intact, maintaining strong co-evolutionary links are maintained in the face of high rates of horizontal transfer. We are currently finalising the details of this analysis.
Exploitation Route The data resulting from this BBSRC project represent a novelty as they are the first direct demonstration of changes in gene expression driven by adenine methylation by restriction modification systems and are a great step forward in the analysis of epigenetic gene regulation in bacteria.

We have developed an app on phase variation with the purpose to educate and engage the public and young people on the role of epigenetics in bacterial adaptation to the human body. 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". The app has been presented so far at three events, two of which at the University of Leicester, including the Dynamic DNA day aimed at schools. Feedback form the students was very positive and has been constructive in planning a full version of the game that should be downloadable for the app store.

We have published a review on the phase variable restriction systems in the high impact factor journal FEMS Microbiology Reviews (Impact Factor: 11.4) which describes the state of the art knowledge on these systems so far (De Ste Croix et al., FEMS Microbiol. Rev. 2017. 41(Supp_1):S3-S15. doi: 10.1093/femsre/fux025). The work on the phase variable type I system SpnIV was also published in the high impact journal Nucleic Acid Research (11.5). This work describes the mechanism of recombination in the system and the effect on horizontal gene transfer (Kwun et al., Nucleic Acids Res. 2018 Nov 30;46(21):11438-11453. doi: 10.1093/nar/gky906.). We plan to submit six further papers in the coming months on the final findings of the paper. The occurrence of these phase variable systems in periodontal pathogens has been explored and the genome sequences published (Haigh et al., Genome Announc. 2017 Nov 16;5(46). pii: e01229-17. doi: 10.1128/genomeA.01229-17.).

A collaboration was set up with the international company Chr Hansen (Horsholm, Denmark) producing food starters and functional food. This contract led to the funding of a KTN CASE BBSRC studentship BB/P504737/1 entitled "Epigenetic control of strain fitness of starter cultures and probiotics". This studentship stated in 2016 and will explore the same type of phase variable methylation system in bacteria of the genus Lactobacillus. Potential outcomes of industrial interest are related to fitness of starter cultures and methods to monitor that fitness.
Sectors Agriculture, Food and Drink,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Work of this grant oh phase variable methylation allowed to get in contact with an Industry producing phage to prevent Listeria infection on food products and to set up a collaboration with a global biotechnology company involved in food starter production. These contacts have produced CDAs, MTAs and a collaboration agreement resulting in direct and in-kind funding to the University of Leicester. With our discovery of intracellular replication of pneumococci we were able to successfully apply for a CASE studentship with GSK and are now again planning to apply with GSK for a BactiVAC Network grant in May.In both cases the aim is to propose our data on within-macrophage survival as correlates of protection.
Sector Agriculture, Food and Drink,Healthcare
Impact Types Economic

 
Description CASE studentship
Amount £99,000 (GBP)
Funding ID BB/S507052/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description CASE studentship
Amount £36,000 (GBP)
Funding ID BB/S507052/1 
Organisation GlaxoSmithKline (GSK) 
Sector Private
Country Global
Start 10/2018 
End 09/2022
 
Description ESCMID study group grant
Amount € 30,000 (EUR)
Organisation European Society of Clinical Microbiology and Infectious Diseases (ESCMID) 
Sector Charity/Non Profit
Country Switzerland
Start 03/2016 
End 12/2017
 
Description High Speed super-resolution confocal laser scanning microscope for sub-diffraction analysis at the multi-user Leicester Advanced Imaging Facility
Amount £283,824 (GBP)
Funding ID BB/S019510/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2019 
End 06/2020
 
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 Linking bacterial sugar metabolism and cell-to-cell signalling
Amount $399,000 (AUD)
Funding ID DP190102980 
Organisation Australian Research Council 
Sector Public
Country Australia
Start 04/2019 
End 03/2022
 
Description Multi-user Equipment Grants
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
 
Description Royal Society Wolfson Refurbishment Scheme
Amount £75,000 (GBP)
Funding ID WL160026 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 09/2017
 
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 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 Listeria monocytogenes phase variable hsdS alleles 
Description In any given wild type population of Listeria monocytogenes CC4, CC5, CC8, and CC54 a Type I Restriction Methylation System can be found to exist in four different allelic forms. This work describes a protocol for allele quantification for these allelic forms in CC4 strains. the protocol 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 four allelic variants; this is followed by a digestion with EcoRV giving rise to four 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 for hsdS alleles. Using this protocol we are able to determine the hsdS allele composition of any CC4 L. monocytogenes strain in less than 3 days. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact the toll allows monitoring of sample composition and provides an important tool for validation of htgs attempts to quantify allele prevalence in a given sample. 
 
Title Quantifying Streptococcus pneumoniae for the six-alleles system SpnD39IV TVR 
Description In any given wild type population of Streptococcus pneumoniae the Type I Restriction Methylation System (SpnD39IV or TVR) can be found to exist in four different allelic forms. This work describes a protocol for allele quantification for these SpnD39IV 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 four 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 four 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 SpnD39IV forms. Using this protocol we are able to determine the SpnD39IV allele composition of any pneumococcal sample in less than 3 days. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
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 Epigenetic control of meningeal pathogenicity of Listeria monocytogenes 
Organisation University of Bern
Country Switzerland 
Sector Academic/University 
PI Contribution we have provided the University of Bern with confidential data on phase variable methylation in Listeria and discussed a collaboration on the meningeal virulence of this organism. We in addition provided the group with information on pneumococcal and Listeria replication in splenic macrophages and enquired the set up of an organotypic culture system for the spleen
Collaborator Contribution the partner has provided us with a first set of clinical isolates including clinical data and tested the feasibility of an organotypic culture for spleen slices.
Impact this collaboration which was significantly boosted by being awarded the BBSRC grant has led to a joint application for funding by the European Society of Clinical Microbiology and Infectious Diseases ESCMID. being both part of the ESCMID study group of infections of the brain we applied for a ESCMID study group grant in September 2015; which was successful. A manuscript is being drafted.
Start Year 2015
 
Description Epigenetics Mobile Application 
Organisation Game Dr Limited
Country United Kingdom 
Sector Private 
PI Contribution discussion on how to design a downloadable app to teacht phase variation to school students
Collaborator Contribution expertise in game design
Impact The app is available and has been used in training events at the University of Leicester and in other institutions. A fully functional version is planned to be freely available for download on phones and ipads. we are finalising a mansucript : De Ste Croix M, C Brown, MR Oggioni. Epigenetics Mobile Application: a mobile game app to teaching phase variation in infectious disease. To be submitted to Journal of Biological Education.
Start Year 2017
 
Description Impact of phase variable methylation on resistance of Listeria populations to phage infection 
Organisation Micreos
Department Micreos Food Safety BV
Country Netherlands 
Sector Private 
PI Contribution A confidentiality agreement and MTA is being finalised with Micreos, a Dutch company producing and marketing a bacteriophage for use in food processing and handling to prevent Listeria growth and human infection. The collaboration is aimed to test the effect of phase variable restriction on phage resistance of a bacterial population. The role of my group is to test population dynamics of bacteria with phase variable methylation systems infected with phages having different methylation profiles.
Collaborator Contribution the industrial partner will share expertise on phage production licensed for use on food and food processing and handling
Impact the collaboration is aimed to provide know how and methodologies for industrial phage production
Start Year 2016
 
Description Large scale genomic screening of Listeria monocytogenes strains for phase variable genes 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution we have provided the University of Zurich with confidential data on phase variable restriction systems in Listeria and detailed initial observations on clonal complex specific distribution and offered a collaboration on the topic
Collaborator Contribution The partners have performed PCR on 250 bacterial isolates for three distinct restriction modification systems, performed genome sequences of four isolates and provided methylome data for other 10 isolates. Presently the partners are preparing DNA from 250 bacterial isolates for genome sequencing.
Impact Raw sequence files and draft genomes in contig level for all 160 Swiss isolates were submitted to the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under study accession number PRJNA486730. A manuscript darft has been submitted: Zamudio R, JD. Ralph, RD Haigh, M De Ste Croix, T Tasara, K Zurfluh, MJ Kwun, SD Bentley, NJ Croucher, R Stephan, MR. Oggioni. Detection of lineage specific evolution in Listeria monocytogenes through the genome analysis of a panel of Swiss isolates from food and human origin. Submitted.
Start Year 2015
 
Description Phase variable epigenic control in food bacteria 
Organisation Chr. Hansen A/S
Country Denmark 
Sector Private 
PI Contribution A KTN CASE BBSRC PhD studentship was obtained in collaboration with Chr Hansen as they are interested in the potential industrial relevance of phase variable epigenetic gene control on starter culture fitness. We have identifies phase variable methylation systems in food grade bacteria and are working on their characterisation
Collaborator Contribution Chr Hansen has provided us with genome sequences and strains of industrial relevance and hosted the PhD student for 3 months in their research laboratories in Denmark.
Impact Two manuscripts are in the process of being finalized.
Start Year 2016
 
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
 
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 Epigenetics Mobile Application: a mobile game app to teaching phase variation in infectious disease. 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We have developed together with Game Dr Limited, 24 Oxford Terrace, GL13NT an app which can be downloaded onto mobile devices to play interactively and learn about phase variation. The app was tested at the Outreach event Dynamic DNA of the University of Leicester with great success. Two ipads and the app were paydid on the grant. The was great interest of the school students in the app and folow up discussion with the post doc of the project who lead the activity.
We are planning to fund with other funds the completion of the app so that a fully functional version can be made available for download on the app store.
A publication is planned: De Ste Croix M, C Brown, MR Oggioni. Epigenetics Mobile Application: a mobile game app to teaching phase variation in infectious disease. To be submitted to Journal of Biological Education.
Year(s) Of Engagement Activity 2018,2019