Systematic identification of novel anti-phage defense mechanisms in the E. coli pangenome

Project Summary Title: Systematic identification of novel anti-phage defense mechanisms in the E. coli pangenome There is an urgent need for new therapies and approaches for treating antibiotic-resistant bacterial infections. One promising, but underdeveloped approach called phage therapy aims to use the viruses that infect bacteria, called bacteriophages (or just phages). Although there have been a handful of case studies reporting success, including for the treatment of ESKAPE pathogens of most dire concern, the long-term efficacy and prospects for wide-spread use of phage therapy remains highly uncertain. A key challenge is that bacteria often harbor potent anti-phage defense mechanisms that enable them to resist or overcome viral infection. These anti-phage mechanisms have emerged from the long-standing, fierce coevolutionary battle between bacteria and phages, with a molecular 'arms race' leading bacteria to evolve diverse mechanisms for defending themselves and phages, in turn, evolving counter-defense strategies. The anti-phage arsenal of bacteria includes restriction- modification (RM) and CRISPR-Cas systems. In recent years computational studies have identified dozens of additional systems, but these studies have critical limitations, and our own experimental studies have indicated that there are dozens, and likely hundreds, of additional systems still to be discovered. By developing and applying a powerful, high-throughput functional selection procedure, we aim to identify the anti-phage systems present in a diverse collection of 1,500+ strains of E. coli, including a range of pathogenic strains. We will screen for defense against a panel of 10 different coliphages. Bioinformatic analyses, particularly homology detection and structural predictions, will be done to assess the conservation, genomic context, and predicted biochemical functions of the newly identified systems. Thus, our work will lay the foundation for detailed molecular studies of the diverse new systems identified. As with prior studies of anti-phage defense, we anticipate that the new systems will drive the discovery of new molecular mechanisms, which may, in turn, form the foundation of a new generation of precision molecular tools. It has also become clear in recent years that many cell autonomous components of eukaryotic innate immunity have distant homologs in bacteria. As such, our work may also reveal evolutionarily conserved facets of immunity across the kingdoms of life. Finally, the methodology developed will be broadly applicable to virtually any bacterial pathogen, work that we anticipate will inform ongoing and future efforts to develop phages as therapeutic agents.