Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron Tomography

For successful infection, bacteriophages must overcome multiple barriers in the bacterial cell envelope to translocate viral DNA and proteins into the host cell. However, the molecular mechanisms underlying the phage infection initiation remain poorly understood. Here, we use cryo-electron tomography of Salmonella cells infected by phage P22 to capture intermediates during infection. P22 particle initially attaches to the cell surface obliquely through interactions between phage tail spikes and bacterial lipopolysaccharides. Subsequently the phage needle penetrates the host membrane. Three ejection proteins (gp7, gp16, gp20), which are originally stored in capsid, are ejected and assembled to create a 40 nm long trans-envelope channel between the phage tail and the cytoplasmic membrane. The novel channel serves as the conduit for genome translocation. Using mutant particles lacking one or more of the ejection proteins, our in situ structural analysis demonstrates that gp7 forms an extra-cellular channel, extending from the phage tail to the outer membrane; gp20 forms a mushroom-like structure spanning the periplasm, while gp16 is involved in channel formation in the cytoplasmic membrane. Together with atomic resolution information from mature particles and their proteins, our studies reveal a series of key intermediate infection structures at unprecedented detail. They also unveil a massive remodeling of both the infecting phage and the bacterial cell envelope. The structures we obtain provide the first direct evidence illuminating the functions of the three ejection proteins and their distinct role in phage genome delivery.