High-Throughput Characterization of Host-Bacteriophage Interactions with an Eye Towards Emerging Pathogenic Threats

Bacteriophage are diverse, critical players in the ecological dynamics and structure of microbial communities through the predation of their respective microbial hosts. Due to specific interactions with their microbial hosts, rational combinations of bacteriophage can be deployed to complex communities and eliminate targeted pathogens. Importantly, design principles are emerging for increased efficacy during such treatments – rational phage cocktail design can reduce the risk of pathogen escape and increase population-level antibiotic sensitivity. However, leveraging these design principles into actionable treatments, products, and bottom-up engineering practices relies upon extensive characterization of how the bacteriophages interact with pathogen physiology. Recent work from our group and others demonstrated substantial increases in both the pace and resolution that we can infer bacteriophage-host interactions by use of barcoded, pooled genetic screens. Here, we extend random bar code transposon-site sequencing (RB-TnSeq) to Salmonella typhimurium to investigate thousands of genetic interactions with eleven of its bacteriophage, of which only four had substantial characterization prior to this work. We uncover receptor requirements for all eleven phages tested, including novel, unanticipated, complex receptor requirements that would not have been discovered without multiple, unbiased genetic screens. We additionally discover and characterize new host-side requirements for broad-host range phages, highlighting the importance of intracellular transcription and environmental conditions. This work comprises the largest, functional characterization of Salmonella bacteriophages, providing new avenues, considerations, and features for rational phage cocktail design against this emerging pathogen.