The temperate bacteriophages of Pseudomonas aeruginosa and their role in chronic lung infection

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen common in respiratory infections of both cystic fibrosis (CF) and bronchiectasis (BR) patients and is associated with reduced lung function and poor clinical outcome. Pa is proficient at establishing chronic infections of the lung due to its ability to adapt and evolve under environmental selection. It achieves this, in part, by being naturally competent, pliable and open to horizontal transfer of nucleic acids by conjugative plasmids and bacteriophages (phages) etc. The focus of this research is temperate phages, bacterial viruses that infect and integrate into the chromosome of their host bacteria either being replicated like any other genetic loci or induced to a replicative state that lyses the cell, allowing release for further bacterial infection. In their integrated, prophage state, their diversity and gene carriage offer altered cell function. This work illustrates that temperate phages are extremely common in Pa isolated from the lung, offering diversity that can aid bacterial fitness and virulence through the subversion and modification of bacterial metabolic function. \n \nUsing LC-MS and metabolomics analysis this research demonstrates that each of the Liverpool Epidemic Strain (LES) prophages subvert cell function and alter their Pa host’s metabolism. This was then broadened to include studies focussing on the phages isolated from different aetiologies of chronic respiratory infection to see if these metabolic changes occur within a larger panel of bacteriophages. This determined that whilst there are limitations in aligning function in a high number of metabolites found using this approach, there are phage mediated differences between lysogen and wild-type bacterial host, where a core shift in metabolism was observed in Pa converted with phages from different arms of these studies. Importantly, it shows that temperate phages play an intrinsic role in altering bacterial cell metabolism. \n \nTo complement this work there is focus on the wider genetic diversity of Pa prophages, which allowed a collaboration with Prof. Roger Levesque in Laval, Canada and access to the International Pseudomonas Consortium Database. From >1000 Pa genomes 8 groups of phages clustered by type were determined (e.g. F10-like phage). Lysogens were created in PA14 with individual phages from each (4 out of 8 groups). Host virulence genes across the International Pseudomonas aeruginosa consortium database (IPCD) phages was also investigated illustrating carriage across the panel, something that is not this widespread when reported in other bacterial backgrounds. \n \nThis research identifies possible transfer between Pa morphology variants, also different MLST types, isolated from the same patient at the same timepoint, something that has been hypothesised, but not shown prior to this study. However, this only shows a single timepoint and snapshot time and an important focus must be the longitudinal relationship between bacterium and phage, evoking the well named ‘evolutionary arms race’ and promotion of selection in the chronic lung. This longitudinal relationship was studied by surveying the carriage of Pa prophage in the chronically infected lung of BR patients, which illustrates both genome expansion and reduction in these virus genomes over time. We focus further on this genetic shift/drift in these longitudinal samples. Cross infection of phages induced from these isolates did not offer the host restriction over time we envisaged, which further complexes the story of phage sensitivity over time. \n \nAs a whole, this research demonstrates the complexity temperate bacteriophages add to the diversity of Pa in the chronic lung, influencing adaptation and evolution that promotes continuing colonisation. Lysogens derived from a lab strain of Pa and BR/CF phages illustrated lowered pathogenicity in the Galleria waxmoth larval model compared to the wildtype host. This is a trait that is seen in the CF/BR lung with lowered immunogenicity of Pa in later stages of CF/BR chronic respiratory infection. This work provides the foundations and hypotheses to further explore the phage bacterial relationship in the lung and its role in persistence and disease.

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