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

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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.