Prophage host interactions: pulling back the curtains on Pseudomonas puppet masters

Prophages are viruses (bacteriophages) that have integrated into bacterial genomes, but their contribution to the success of their hosts has been grossly under-estimated. We aim to provide a more detailed view of prophage influence on the bacterial host, which will greatly expand our understanding of a system where they are known to be important, but not how they are important.
Pseudomonas aeruginosa, a pathogen of plants, animals and humans will be our model prophage/bacterial host system. We will specifically focus on the Liverpool Epidemic Strain (LES) and, a less pathogenic model strain, PAO1. The LES strain is known to carry multiple prophages that have been proven to enhance its competitiveness. The LES prophages do not encode any known toxins or virulence factors but have been associated with altered metabolic pathways and other biological traits of P. aeruginosa. Prophage sequences are found in almost all bacterial strains. However, very little is actually known about the genetic information that these prophages carry. In fact 75% or more of most prophage genes are annotated as hypothetical (often referred to as phage dark matter). These hypothetical sequences may be shared across many different phages but, unless they have been identified as virulence-related factors, the relevance of these sequences to the biology of the host is generally ignored. Recently, the importance of some of this dark matter has been elucidated and shown to regulate bacterial host genes or to promote bacterial survival. At this point in time when we are losing the battle in controlling the spread of multidrug resistant bacterial infections, there is an urgent need to better understand all of the genetic elements that are controlling bacterial biology. These data will help to produce better informed strategies for the control of bacterial infection and also aid the understanding of lytic bacteriophages that are currently being used in the development of phage therapy.
Our objectives will uncover the hidden mechanisms by which prophage "puppet masters" affect the biology and fitness of P. aeruginosa: We have purified multiple inducible prophages from the LES strain of P. aeruginosa. We have constructed a precise set of tools and strains to investigate the direct effect of each prophage (separately and in combination) on a well-characterised model host strain, compared to a strain where the prophages are absent. We will use cutting edge techniques, combining knowledge of genome architecture, changes in gene expression and putative regulators to reveal the different ways that prophages impact their bacterial hosts. Cloning of identified regulators and mutant construction will enable association of prophage genes to functional pathways. We will monitor the impact of identified prophage-encoded elements under many varied environmental parameters that reflect the niches of P. aeruginosa. This combined approach will elucidate the interactions between three cohabiting LES prophages and their host to understand better their control of the bacterial behaviour.
Applications and benefits: Not only will these studies inform a better understanding of P. aeruginosa biology, but the techniques can be applied to other phage-host systems. Bioinformatic tools have been much improved in recent years, for identifying prophages in bacterial genomes. But without functional studies such as ours, the relevance of the phage genes remains part of the dark matter. Identifying function of unknown genes (that are conserved across phage databases) will transform the field of phage biology and our fundamental understanding of microbiology.

LES prophages are known to impact the fitness of their lysogen, but lacking any obvious genes to mediate fitness, their importance remains unexplored. We propose to unpick the LES prophages contribution to bacterial fitness and reveal the underappreciated level of control that prophages have over bacterial host functions. Using strategies proven to work in for shigatoxigenic prophages in E. coli, we will first maximise the stability of the prophages during lysogen culture as well as optimise prophage induction conditions. We will use the induction conditions to purify the virions and then use LC-MS/MS to actually identify the prophage genes encoding the structural proteins of the phages. We have produced a comprehensive set of isogenic PAO1 lysogens and designed 36 library conditions from which we will extract total RNA, using 9 strains grown in 2 different media that highlight prophage-mediated phenotypic changes of the host under conditions where the prophages are most stable and under prophage inducing conditions. We will use RNAseq to identify differential gene expression (mRNA and small RNA) as well as identify transcription start sites. The identification of differentially expressed (DE) genes and prophage genes that are expressed from a stable prophage will enable us to build phenotypic assays and prophage gene constructs to test the function of prophage effectors to control the DE profiles of the Pseudomonas host. Having identified prophage effectors of host DE profiles we will then test a variety of strains using Nanostring's nCounter platform to investigate the global nature of our work. These approaches are needed as most phage genomes are comprised of at least 75% "dark matter" (hypothetical or ORFs of unknown function). Understanding the increased fitness of LES will enable better understanding of its biology and will pull back the curtain over mechanisms used by prophages to alter their bacterial hosts.

Understanding the role of prophages in controlling their bacterial hosts will aid our understanding of bacterial gene regulatory networks, prophage impacts on bacterial hosts and our ability to assign function to genes that simply can not be characterised, currently.
1. SCIENTIFIC COMMUNITY: (microbiologists, phage biologists, genomics researchers). By empirical comparison of isogenic strains that differ only in prophage carriage our data will generate a clearer understanding of how prophage contribute to bacterial fitness. This information will aid bacteriologists in better understanding how bacteria respond to different stresses. Prokaryotic genomes are being sequenced and annotated at an increasing rate, but phage annotation is lagging behind (most genes cannot be annotated), which represents a critical gap in our knowledge - especially around potential genetic targets for intervention. A lack of functional annotation obfuscates study and comparison in microbiological, epidemiological or evolutionary studies. The project described here will begin to provide meaningful functional data, which will inform future experimental approaches, leading to new understanding and breakthroughs in academic and applied microbial research.
2. PUBLIC SECTOR: including clinical and agricultural stakeholders (eg CF Foundation Trust, NHS, WHO, DEFRA, OIE , AHDB). Our research is of direct relevance to key questions about how to treat bacterial diseases in agriculture and healthcare, and will therefore potentially improve farming productivity and wealth as well as the health and welfare of managed animals and humans. On a wider scale, elucidation of P. aeruginosa-phage interactions enables similar studies across a range of other bacteria. Whilst some Pseudomonas species are responsible for infections of economically important crops, cattle or humans, other species have industrial or environmental relevance, forming disruptive biofilms in pipelines or applied as bioremediation agents. A better understanding of phage relationships with these organisms could have wide economic impact on industrial processes and environmental management. Phage therapy approaches using phages informed by our data could have a direct and financially tractable approach for tackling antibiotic resistance.
3. PRIVATE SECTOR: pharmaceutical and biotechnology companies. Considering the catastrophic threat of AMR, our research could inform the development of desperately needed therapeutic strategies based upon new knowledge of gene regulation and environmental response, benefitting the pharmaceutical industry and new public-private partnerships that have formed to address this need (eg Novartis, Vertex Pharmaceuticals, GSK, Phizer, AMR Centre). Our data and methods will also be of great interest to phage therapy companies and will springboard the wider elucidation of function for so many poorly annotated phage genes (eg Adaptive Phage Therapeutics). Such elucidation could reveal novel aspects of phage biology for exploitation to develop revolutionary biotechnologies e.g. the history of CRISPR gene (eg Bayer, Blue Rock Therapeutics). Patients: eg those with Cystic fibrosis, particularly those with drug-resistant P. aeruginosa will benefit from new treatments that could be developed in response to our new understanding of prophage-regulated P. aeruginosa phenotypes. In the longer term, our data will inspire similar research to identify further therapeutic targets, benefiting patients suffering from other bacterial infections.
4. GENERAL PUBLIC AND MEDIA: (eg science festival goers, those with an interest in infection and societal impact of microorganisms). Antibiotic resistance is a global, catastrophic threat, and stories about bacteriophages, conceptually and as therapeutic tools, are increasingly on the public and media radar. Engagement offers the chance to educate and inspire interest in phages, infections, novel treatments and STEM subjects in general.