Illuminating jumbo phage infection mechanisms: from complex prokaryotic cell biology to novel therapeutics

About This Grant

Project Summery/Abstract Amid a public health crisis driven by antibiotic-resistant pathogenic bacteria, bacteriophages (phages), which naturally infect and kill bacteria, represent a promising alternative as antimicrobials. However, a significant challenge is posed by diverse bacterial immune mechanisms that resist phage infections. Overcoming this obstacle requires phages equipped with robust anti-immune capabilities. In this context, ΦKZ-like jumbophages (genomes > 200kb) have an exceptional ability to counter various bacterial nucleolytic immune systems throughout infection, with numerous family members targeting key Gram-negative pathogens. The jumbophage ΦKZ is a broad host range killer of the multi-antibiotic-resistant pathogen Pseudomonas aeruginosa and serves as the leading model phage for this family. Immune evasion is largely achieved through the assembly of a bacterial membrane lipid derived compartment termed the “Early Phage Infection Vesicle” (EPIV), which I co-discovered during my postdoctoral work, and a phage-encoded proteinaceous compartment called the “phage nucleus,” which shields the replicating phage genome. The long term goal of this study is to understand three unexplored aspects of jumbophage biology related to the biogenesis and functioning of the EPIV. The EPIV houses early transcription, but the phage has to solve a fundamental challenge not previously solved in bacteria–mRNA export from a lipid-bound compartment and successful docking with ribosomes, which are unusually uncoupled from transcription in this case. I hypothesize that a novel mRNA export channel, analogous to the eukaryotic nuclear pore complex, is assembled by injected ΦKZ proteins to export mRNA to ribosomes. I will uncover this complex using cryo-ET, mass spectrometry and genetics. I will additionally examine the role of EPIV assembly in enabling ‘pseudolysogeny’ in jumbophage infections. This process of phage quiescence was observed in jumbophages long ago but lacks a mechanistic understanding. I hypothesize that the EPIV has the potential to be a stable pseudolysogenic entity inside an infected bacterium that I will test herein with my multidisciplinary approach. Finally, I will attempt to elucidate the mechanism of EPIV biogenesis – it remains entirely unknown how this massive membrane-bound organelle is rapidly assembled within bacteria and how its formation is conserved across diverse jumbophage infections. To answer these questions, I will combine genetic dissection and c-ET to reveal the key participants and early assembly events of this unique phage-driven prokaryotic organelle formation. Overall, my studies stand to uncover fundamentally fascinating bacterial-phage cell biology in addition to innovative and potentially transferable mechanisms to enhance phage success in combating pathogenic bacteria. This research will be conducted at UCSF, which hosts state-of-the-art facilities and a highly intellectual and collaborative research community. It will also provide me with the training in genetics and structural biology that I need to fulfill my postdoctoral training goals and pioneer an independent research program in bacterial-phage interactions.