Genetic and Proteomic Approaches to Reveal Bacterial Vulnerabilities to Phage Predation

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PROJECT SUMMARY We are entering a post antibiotic world where understanding the mechanism of an antibacterial strategy is not sufficient to ensure an effective therapy. We must also consider the mechanisms of resistance and address them during the design process. We will use genetics and proteomics to discover how bacteria combat bacteriophage (phage) lysis. Driving this goal is the desire to combat phage resistance mechanisms so as to make bacteria more susceptible to phage predation. We first tackle the problem by employing state of the art genetic methods to interrogate the role of essential genes in limiting phage replication and bacterial lysis. Using CRISPR transcriptional interference (CRISPRi), we will conduct the first studies in the human pathogen Pseudomonas aeruginosa to determine whether partial knockdown of essential genes can positively impact phage replication. We hypothesize that inhibition of certain essential genes will not only limit bacterial fitness, but also has the potential to enhance the success of any phage-host pairing, regardless of whether the a priori state is one of phage resistance or sensitivity. Put another way, even phages that already replicate in a given host can do better. We will additionally harness the ease of CRISPRi screening to identify non-essential genes that limit phage replication in strain-dependent manners, more akin to canonical hypervariable immune systems (e.g. CRISPR). To further our understanding of the physical underpinnings of phage resistance, we will create a physical map of phage-host protein-protein interactions using whole cell fractionation proteomics. This is particularly critical as many phage proteins are of unknown function and is in line with our goal of identifying essential protein complexes interacting with phage factors. We will validate the importance of factors identified from genetic and proteomic assays with phage replication assays during single gene knockdown to assess generalities of phenomena observed. Lastly, we suspect that phage “accessory genes” (i.e. hypervariable loci not strictly essential for phage replication in all hosts) represent a treasure trove of inhibitors and modulators of host processes, which could be useful genetic fodder for enhancing future phage therapeutics. Accessory genes have been bioinformatically identified and host binding partners will be identified with conventional affinity purification- mass spectrometry, and validated with phage replication assays. Our research strategy combines the complementary expertise of three investigators: Joseph Bondy-Denomy, an expert in phage biology and bacterial immune systems; and Danielle Swaney, an expert in bioanalytical mass spectrometry who has used her skills to define the host-pathogen protein-protein network for many human pathogens; and Jason Peters, a microbiologist with expertise in bacterial genetics and pathogenesis.