Elucidation of the mechanisms of jumbophage genome protection during infection

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In the face of a public health crisis of pathogenic bacteria resistant to most existing antibiotics, bacteriophages (bacterial viruses; phages) that naturally infect and kill bacteria, represent a promising alternative for use as antimicrobials. However, a significant obstacle to realizing this translational goal is presented by the plethora of immune mechanisms in bacteria to resist phage infections. Overcoming this obstacle necessitates phages equipped with robust anti-immune capabilities to kill multiple isolates of antibiotic-resistant pathogenic bacteria. In that regard ΦKZ-like jumbophages (phages with genomes > 200kb) emerge as a family with an outstanding ability to thwart a multitude of bacterial nucleolytic immune systems throughout infection and numerous family members that infect most important Gram negative pathogens. The jumbophage ΦKZ is a broad-host range killer of the multi-antibiotic resistant pathogenic bacteria Pseudomonas aeruginosa and the leading model phage for the jumbophage family. Immune evasion is achieved largely through the assembly of a phage-encoded proteinaceous nucleus-like “shell “compartment that encapsulates and shields the replicating phage genome. However, it is unknown how this phage protects its genome prior to the assembly of the shell. The long term objective of this proposal is to understand the mechanisms used to guard the ejected genome of ФKZ-like jumbophages–preliminary evidence (from us and others) suggests the assembly of a macromolecular compartment in infected cells at similar locations as the ejected jumbophage genome (prior to shell assembly). Currently, the composition and biological significance of these complexes remain unknown. I hypothesize that this complex (termed herein the ‘ejected structure’) creates a DNA-containing organelle that encapsulates and protects the ejected phage genome from immune nucleases. I will first use fluorescence microscopy and cryo-electron tomography to visualize the infection state prior to shell assembly. From these studies I will gain high resolution structural insight into the biogenesis and organization of these compartments. In parallel, I will use mass spectrometry to reveal the constituents of these assemblies and subsequently interrogate the functional roles of these constituents in supporting phage infection. From my multidisciplinary studies, I stand to uncover fundamentally new phage biology in addition to innovative and potentially transferable mechanisms to enhance phage success in the fight against pathogenic bacteria. I will perform this work at UCSF, an institute that hosts state-of-the-art research facilities and fosters an intellectually rigorous and collaborative research environment. Co-supervision in the Bondy-Denomy and Agard labs will ensure thorough training in phage biology, genetics and biophysics. Through the course of my research, I will undergo extensive training and subsequently gain expertise in diverse fields of microbiology, molecular biology, biophysics and structural biology that I need to fulfill my postdoctoral training goals and subsequently pioneer an independent research program.