Visualizing the structural changes of bacteriophage Epsilon15 and its Salmonella host during infection

Abstract

The efficient mechanism by which double-stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single-particle electron cryomicroscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (ɛ15) and P-SSP7. We have used electron cryotomography and three-dimensional subvolume averaging to capture snapshots of ɛ15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of ɛ15 attach to the surface of the host cell. Next, ɛ15's tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.

Figure 1. Electron cryo-tomography

(a) A slice through the 3D reconstruction of bacteriophage ε15 incubated with resting cells shows full capsids attached to the periphery of the cell. (b) A slice through the 3D of ε15 incubated with cells at log phase shows empty capsids around the cell. (c) A slice through the 3D shows full (vertical arrow) and empty capsids (horizontal arrow) attached to ghost cells (bacterial membranes). Bar is 500 nm.

Figure 2. Bacteriophage ε15 in isolation

(a) On the left, a slice through the center of the single-particle map shows the tailspikes, tail hub, portal, core, capsid, DNA shells, and DNA segment . On the right, these components are annotated in color (reproduced from Jiang et al ). (b) On the left, a slice through the capsid position labeled in Figure 2a shows the DNA segment (bright dot in the center), surrounding portal, DNA shells around the portal, and capsid. On the right, these components are pseudo-colored. (c) On the left, a slice through the labeled tail position shows the closed tail hub as a 6-sided star surrounded by 6 tailspikes. On the right, these components are pseudo-colored. (d) A slice through the center of the map derived from subvolume averaging shows the tailspikes, tail hub, and full capsid, but the portal, core, and DNA can not be resolved. (e) A slice through the labeled capsid position shows a filled capsid. (f) A slice through the labeled tail position shows the tail hub as a solid density with 6-fold characteristic and surrounded by 6 tailspikes.

Figure 3. Subvolume averaging of full bacteriophages

(a) A slice through the center of the map of bacteriophages attached to ghost cells shows the tailspikes, tail hub, full capsid, and indented cellular membrane (the bowl below the tail, arrow), but the tail hub is not connected to the cell membrane. (b) A slice through the capsid position labeled in Figure 3a shows a punctate but mostly solid density at the portal location. (c) A slice through the labeled tail position shows the tail hub having 6-fold characteristics and surrounded by 6 tailspikes. (d) A slice through the center of the map of bacteriophages attached to resting cells shows the tailspikes, tail hub, full capsid, and cell surface (horizontal density beneath). The tail hub density is continuous with and attached to the cell. (e) A slice through the capsid position labeled in Figure 3d shows the portal and DNA as punctate density. (f) A slice through the labeled tail position shows the tail hub surrounded by tailspikes.

Figure 4. Subvolume averaging of empty bacteriophages

(a) A slice through the center of the map of bacteriophages attached to ghost cells shows tailspikes, tail hub with hollow interior, empty capsid (absence of core and DNA), cellular membrane, and a tubular density in the periplasmic region. Vertical bar is 21 nm tall. (b) A slice through the capsid position labeled in Figure 4a shows the portal as a hollow ring and absence of DNA. (c) A slice through the labeled tail position shows faint densities corresponding to a hollow tail hub and tailspikes. (d) A slice through the center of the map of bacteriophages attached to cells shows tailspikes, tail hub with hollow interior, empty capsid, cell surface, and tapered density in the periplasmic region. (e) A slice through the capsid position labeled in Figure 4d shows the portal as a hollow ring. (f) A slice through the labeled tail position shows a hollow tail hub surrounded by 6 tailspikes.

Figure 5. Infection model

(a) Free-floating bacteriophage tailspikes come into contact with and recognize an LPS on the cell surface. (b) The virus digests the LPS to presumably reach the cell surface and (c) binds to a receptor on the cell outer membrane, causing the tail hub to open. (d) The core exits the capsids and forms a tube across the periplasmic space, thereby directing the viral DNA into the host cell cytoplasm. (e) After the viral genome has been completely internalized, the perisplamic tube seals to prevent leakage of cytoplasm, while the empty capsid remains on the cell surface. Key: lipopolysaccharides (LPS), outer membrane (OM), peptidoglycan layer (PL), inner membrane (IM).