The role of prophages in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common opportunistic respiratory pathogen of individuals with cystic fibrosis (CF), capable of establishing chronic infections in which the bacterial population undergoes extensive phenotypic and genetic diversification. The Liverpool Epidemic Strain (LES) is a widespread hypervirulent and transmissible strain that is capable of superinfection and is linked to increased morbidity and mortality, relative to other P. aeruginosa strains. The LES has six prophages (LESφ1-6) within its genome, of which three are essential to the competitiveness of this strain. Temperate bacteriophages are incredibly common in bacterial pathogens and can contribute to bacterial fitness and virulence through the carriage of additional genes or modification of existing bacterial genes, lysis of competitors, or by conferring resistance to phage superinfection. Furthermore, the LES phages are detected at high levels in the CF lungs and have been implicated in controlling bacterial densities. The aims of this study were to (i) further characterise the LES phages and their induction, (ii) determine the extent to which the LES phages contribute to bacterial phenotypic and (iii) genetic diversification and (iv) determine how the LES phages affect host competitiveness, using a variety of in vitro and in vivo infection models. LES phages are continuously produced by spontaneous lysis and this study found that environmental factors that are common to the CF lung, such as oxidative stress, pharmaceutical chelating agents and antibiotics, can alter phage production by clinical LES isolates. Characterisation of the phages highlighted differences between the phages with regards to their lytic cycles and ability to propagate in different environments. P. aeruginosa undergoes extensive phenotypic diversification in an artificial sputum model (ASM) of infection, similar to that observed in chronic CF infections. Hypermutability, loss of motility and auxotrophy were phenotypes observed in bacteria evolved for approximately 240 bacterial generations in ASM in the presence and absence of the LES phages. However, the LES phages accelerated this process; loss of twitching motility occured earlier in populations evolved in the presence of phages. Sequencing of evolved populations revealed a high level of genetic diversification, with genes involved in motility, quorum sensing and genetic regulation experiencing loss of function mutations in parallel populations. In phage treated populations, LESφ4 had disruptively integrated into motility and quorum sensing genes, suggesting that temperate phages can provide an alternative (and quicker) route to adaptation. LES prophage carriage is important for bacterial competitiveness; PAO1 LES Phage Lysogens (PLPLs) successfully invaded a phage-susceptible population in vitro from when initially rare. Strain invasiveness was dependent on the LES prophage; LESφ4 lysogens were more invasive than PLPLφ2 or PLPLφ3, whereas carriage of all three prophages accelerated bacterial invasion. PLPLφtriple could also invade a susceptible competitor population in a rat model of chronic lung infection, although not as successfully as in vitro. These data suggest that prophage carriage is important for LES competitiveness and that phage-mediated lysis of phage-susceptible competitors may explain why LES is adept at superinfection. The study indicates that the LES phages are important drivers of bacterial diversification and evolution and confer a competitive advantage to their bacterial host. This may help explain why the LES is so successful, and the high prevalence of polylysogeny in bacterial pathogens.