Functional prophage and lysogen engineering in Citrobacter enabling studies of virulence and other traits

Gut infections due to bacteria are common and problematic and they can cause minor disabling diseases to very serious - indeed lethal - infections. E. coli is one pathogenic bacterium that regularly hits the news headlines where particular strains cause various intestinal diseases ranging from "travellers diarrhoea", and enteropathogenic E. coli disease (EPEC) to enterohaemolytic E coli disease (EHEC) that can lead to kidney failure. The microbiology and molecular biology of some aspects of these diseases are characterised but there can be problems in research progress where an animal model of the relevant infection is not available. This is an issue for EPEC and EHEC where infections of mice are not easy or reproducible. However, some years ago a natural variant strain of a related bacterium (Citrobacter) arose in domesticated laboratory mice and this strain (subsequently classified as Citrobacter rodentium) showed many of the clinical traits of the human pathogens such as EHEC and EPEC, but in the laboratory mouse - an experimentally exploitable system. Consequently, the bacterium C. rodentium has become a useful surrogate model in the mouse for some human intestinal diseases and disorders, including EPEC and EHEC but also with collateral implications for ulcerative colitis, Crohn's disease, and even research on aspects of colonic cancers.

One step towards understanding C. rodentium as a model organism, and thus the E coli pathogens it models, arose with genome sequencing of C. rodentium which revealed that C. rodentium, EPEC and EHEC may have converged on a common host infection strategy partly through mobile genetic elements ("jumping genes") that enable genomic flux and movement of virulence factor genes. Some of this genomic flux was thought to be driven by prophages (where the DNA encoding production of bacterial viruses is embedded in the bacterial chromosome). Prophage genes can cause "phage conversion" or "lysogenic conversion" where genes carried by the phage can impact the physiology and virulence of the pathogen. Indeed some prophages are actually the main cause of virulence in some bacterial pathogens. Some limited progress has been made on the analysis of the prophages of Citrobacter, but the precise natures of these prophages and their impacts on the physiology, virulence and other traits of the pathogen are not clear. This pump-priming study will further characterise prophages of Citrobacter. Specific regions of the chromosome suggest that they may encode fully functional bacterial viruses (phages) and we recently confirmed this hypothesis in our discovery of a new fully functional prophage in the pathogen. Other regions of the chromosome suggest remnants of degraded and incomplete viral DNA. In this project we will make mutants of Citrobacter in which two fully functional prophage loci are either surgically removed or carry deletions in specific genes found within the prophages (using genetic engineering / editing methods). Banks of these prophage deletion and precise mutants will be characterised to define the impacts of the corresponding mutations on pathogen behaviour. These studies will include the use of simple lab-based microscopic worms for virulence assays (avoiding at this stage the, arguably unethical, use of many laboratory mice for initial virulence screening assays). Other bioassays will include physiological assessments of the C. rodentium mutants, including antibiotic resistance and biofilm studies.

In this way, an interrogation and judicious exploitation of the natural prophages of C. rodentium will tell us about the possible biological impacts of these endogenous viruses in driving or modulating multiple traits of this important model enteric pathogen. Furthermore, in the future, this work should provide a platform for studies on the modification of Citrobacter gut pathogens, or E. coli commensals, through lysogenic conversion due to intelligently engineered prophages.

Virulence and other physiological traits of some pathogenic bacteria can be entirely due to, or modulated by, their resident prophages in a process called "phage conversion" or "lysogenic conversion". This conversion phenotype can be due to the inactivation or aberrant expression of specific host chromosomal genes at the prophage DNA insertion site or due to expression of particular "cargo" genes carried by the prophage in lysogens. There are some well known examples of lysogenic conversion in both Gram-positive and Gram-negative pathogens, including Corynebacterium, Clostridium, Vibrio, and E. coli.

The pathogen, Citrobacter rodentium, is the agent of murine enteric infection that has been viewed as an experimentally-tractable model in the laboratory mouse for some E. coli gut infections due to enteropathogenic E. coli (EPEC) or enterohaemolytic E. coli (EHEC). A few years ago genomic analysis of C. rodentium revealed several chromosomal loci that housed prophage DNA - either as remnants of defective phage genomes or as candidate functional phages. We previously identified one fully functional prophage (phiNP) and recently we reported a "new" fully functional prophage (phiSM). Both of these phages are liberated into C. rodentium culture supernatants and both can adsorb to specific LPS components, infect, lysogenise and replicate in some E. coli strains, thereby displaying some capacity for host promiscuity. Based on our bioinformatic interrogation of the respective phage genomes, the viral DNA integration loci, and wider genomic comparisons we hypothesised that these phages may have impacted the evolution of Citrobacter to virulence through lysogenic conversion. In this short pump-priming project we intend to test this hypothesis, and the notion that lysogenic conversion through either, or both, of these heteroimmune prophages modulates the behaviour of the pathogen with respect to biofilm formation, antibiotic resistance or environmental stresses.

Who will benefit?
This project covers temperate phage virology, bacterial virulence and modulation of bacterial behaviour through phage conversion. It has implications for scientists in universities, institutes and biotechnology industries with interests in pathogenesis and resilience, phage-driven evolution, phage SynBio and potential therapeutics. The project impacts on horizontal gene transfer, antibiotic resistance, biofilms, and dissemination of virulence and fitness genes in bacteria.

The study extends our recent investigations about prophages in a murine enteric pathogen. At this early stage, we have no formal industrial collaborations associated with this particular project (although we have had confidential discussions and collaboration with a US-based biotechnology company with mutual interest in Citrobacter bacteriophages). Historically, our interests in Citrobacter phages evolved naturally, initially through a BBSRC-funded PhD student over 10 years ago, subsequently reinforced through postdoctoral collaborations with PIs at the WT Sanger Institute. There are now multiple academic groups in the UK working with Citrobacter and related enteric pathogens, such as EPEC and EHEC. We hope that our work may enable future collaborative synergies with other groups who have expertise in animal pathogenesis, disease models and the potential exploitation of phages for gut microbiome modulation through SynBio strategies.

How will they benefit?
Our research will be published in quality international microbiological journals. Furthermore, we will disseminate our results in the public domain at an international symposium (e.g. ASM) and a domestic meeting in Edinburgh in 2020. The practical "products" from this work (e.g. engineered strains and phages) will be freely available to researchers who consider these outputs to have utility for their own research programmes.

What will be done to ensure that they have the opportunity to benefit?
Generally, bacterial strains, phages and associated experimental protocols are requested by domestic and international researchers on an ad hoc basis after presentation of results in the public domain. I also have a track record of collaborations, advisory roles and consultancies with UK (and USA) companies over the past 30 years and have filed patents, and supervised multiple BBSRC and CASE students since the 1980s. I appreciate the importance of industrial collaborations as routes to technical innovation and exploitable new knowledge for the UK, so I am keen to foster any industrial collaborations where possible. We will use any opportunities for IP protection arising from this project, with professional advice taken from Cambridge Enterprise. Although we have had interest from a USA biotechnology company, we are very enthusiastic about any domestic biotechnology company or SME involvement.

In the past 15-20 years I have had diverse governance and advisory roles in research institutes, learned societies, and with policy makers, including advising the CSA of Scotland and the Scottish Government through the SSAC. I have also engaged with policy makers in meetings at the House of Lords/Scottish Parliament on antibiotic resistance and the impacts of microbes. This has been generally through my continuing involvement with Learned Society governance roles.

We will continue to "evangelise" about phages in local schools describing how basic research drove innovation by creating translational opportunities for modern industrial biotechnology industries. This project will extend training opportunities for Dr Rita Monson in bacteriology, pathology, virology and physiology. She has excellent talents in science communication and is proactive in outreach engagement. This project provides opportunities to hone her talents in science education. This experience, coupled with university courses on career development, will extend her future employment opportunities.