Investigating the mechanisms that make jumbophages impervious to bacterial immune systems

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Project Summary The viruses that infect bacteria, called bacteriophages (phages), are robust killers. In response to the frequent threat of phage infection, bacteria have developed a suite of anti-phage immune mechanisms, such as restriction-modification and CRISPR-Cas enzymes. Phages have emerged as promising alternatives to antibiotics in our current “superbug” crisis, but immune systems are a barrier for successful phage replication. Broad-spectrum phages that evade immune detection and kill multiple isolates of antibiotic-resistant pathogens may prove essential in this fight. We screened 12 obligately lytic phages infecting the prominent antibiotic- resistant pathogen Pseudomonas aeruginosa to identify phages with the ability to broadly evade DNA-targeting immune systems CRISPR-Cas and restriction enzymes. Jumbophage ΦKZ evaded all six DNA-targeting systems tested, making it the strongest “anti-immune” phage identified to date. The mechanisms behind pan- immune evasion for this phage family will be investigated here, with the goal of making fundamental discoveries at the phage host-interface that could benefit phage therapies and other biotechnologies in the future. ΦKZ is a jumbophage with a 280 kb genome, has many relatives that infect other Gram negative pathogens, and is outstanding in its ability to evade bacterial nucleolytic immune systems. Immune evasion is enabled by the assembly of a phage-encoded proteinaceous nucleus-like shell (“phage nucleus”) that serves as a replicative compartment. However, it is unknown how this phage protects its genome prior to the phage nucleus being assembled and subsequently, how protein inclusion/exclusion is regulated. We have identified phage proteins that are ejected with the genome and hypothesize that an “injected structure” (IS) creates a DNA- containing organelle that occludes immune nucleases. Understanding how ejected proteins can rapidly shield DNA from numerous host nucleases, and how the host fights back against the IS with novel immune systems likely represents fundamentally new phage-host interaction paradigms. Next, the nascent phage nucleus assembles adjacent to the IS and receives the phage genome. Subsequently, the phage nucleus imports proteins involved in DNA replication and transcription, while excluding immune nucleases, through unknown mechanisms. A genetic screen in our lab has identified the first phage mutants defective in protein import, with mutations in a single gene. Structure predictions suggest that the encoded protein may be homologous to the conserved TRPV family of ion channels. We will determine its subcellular localization, interaction partners and in vitro properties with the goal of elucidating how a “nuclear pore” could work in a phage. In sum, our work here will unveil new phage biology driven by the co-evolution of host and virus, leading to innovative and potentially transferrable mechanisms for enhancing phage success in the fight against deadly pathogens.