Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation

Abstract

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail (Siphoviridae) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.

Fig. 1.. Structure of T5 tail tip before and after interaction with FhuA in nanodisc.

(A) Negative stain EM image of phage T5, with the tail tip circled. (B) Scheme of T5 tail tip with the assignment of the different proteins and their copy number. (C) Cryo-EM structure of T5 upper tail tip at 3.5-Å resolution. Left: Isosurface view of the map seen from the side; middle: central slice side view; right: ribbon representation of the modeled proteins. The twist and rise between each ring are noted. (D) Cryo-EM structure of the central fiber at 4.2-Å resolution. Left: Isosurface view of the map seen from the side; right: ribbon representation of the modeled proteins. (E) Map of T5 tail structural proteins and genes. (F) Negative stain EM image of a T5 tail interacting with a FhuA nanodisc (black arrows). The white arrow points to the empty-filled limit of the tail (see also extended data Fig. 1F). The gray arrowhead points to a density going through the nanodisc. (G) Scheme of T5 tip after interaction with FhuA (Tip-FhuA). (H) Cryo-EM structure of Tip-FhuA at resolutions ranging from 3.6- to 4.3-Å resolution. Isosurface view of a Tip-FhuA composite map seen from the side (left), a central slice side view of it (middle top), and a top view (middle bottom). This composite map is formed by the addition of Tip-FhuA C3 open tube and C1 bent fiber maps and is only for visualization purposes; right: ribbon representation of the modeled proteins. The color code in (C), (D), (G), and (H) is the same as in (B). Unattributed densities are in white. Scale bars, 50 nm.

Fig. 2.. Structure of T5 Tip collar and Tip-FhuA complex.

(A) Left: Isosurface side view of the tip common core cryo-EM map at high contour level, centered on the p132 collar (boxed in the inset scheme of the tip). Right: Ribbon representation of a central slice of the collar. The star points to loop 52-60, and the density attributed to LTF_pb1 is in transparent isosurface representation. (B) Left: Isosurface bottom view of the map in (A), slice at the p132 collar level. Right: Bottom view of the four p132 monomers that are not related by the C3 symmetry (colored from light pink to violet and numbered). They interact with two p140 monomers (cyan and blue) and with LTF_pb1 (transparent densities). The pink/black arrows point to the direction of the LTF_pb1, and the N and C termini of the proteins are, respectively, indicated by black dots and asterisks. (C) Isosurface view at high contour level of Tip-FhuA unmasked and unfiltered cryo-EM map, side view (left) and slice (right). The red arrow points to one of the β-hairpin “leg.” The blue arrow points to the protrusion going through the nanodisc (ND). (D) Isosurface view at a lower contour level of Tip-FhuA unmasked cryo-EM map, after a 15-Å low-pass filtering, slice (left) and view from beneath the nanodisc (right). The color code is the same as in Fig. 1. Unattributed densities in (C) and (D) are in white.

Fig. 3.. BHP_pb3 closing and opening of the tube.

(A) Left: Two side views of a BHP_pb3 monomer in ribbon representation, with hdI to hdIV domains colored blue, green, orange, and yellow, respectively; the hdII insertion in cyan; the plug in brown; and the C terminus extension, comprising the hdIV-FNIII linker and the two FNIIIs, in red. Right: Side, top, and bottom views of the BHP_pb3 trimer. One monomer is colored as on the left, and the three plug domains are colored brown. In the bottom view, the FNIIIs have been removed for clarity. (B) Central slice through BHP_pb3 cup, boxed in the inset scheme of the tip (yellow; hdIV-FNIII linker, red; plug, brown) highlighting the 35 resolved residues of a TMP_pb2C trimer in different shades of red. Hydrophobic residues of TMP_pb2C, pointing to the center of the coil are represented in sticks. (C) Overlay of BHP_pb3 before (yellow; plug, orange) and after (cyan; plug, blue) opening of the BHP_pb3 cone, after superimposition of the whole tip. Three side views 90° apart and a top view are shown. In the top view, hdI and hdIV have been removed to highlight the pivotal movement of the hdII insertion domain. A red arrow points to the long helix of hdIII that acts as a hinge (see also movie S4). The long linker and the FNIIIs have been removed for clarity. (D) Top and side views of the open BHP_pb3 trimer with the same color code as in (C), and TMP_pb2* 42 C-terminal residues in magenta. TMP_pb2* C termini are indicated (C) as well as the last built residue in N-terminal (T1085, black arrow).

Fig. 4.. Bending of T5 straight fiber.

(A) Structure of Tip-FhuA BHP_pb3, 42 C-terminal TMP_pb2* residues, and pb4 (boxed in the inset scheme of the tip). BHP_pb3 monomers are in gold, orange, and salmon with the hdIV-FNIII linker colored in different shades of gray, pb4 in different shades of green, and TMP_pb2* in red. All proteins are in ribbon representation, except for pb4 spike that is in surface representation. Top: Top view. pb4 N termini (N) and BHP_pb3 C termini (C) are indicated; bottom: side view. (B) Top: Top view of pb4 spike. pb4 monomers are in different shades of green. The hydrophobic residues pointing toward the interior of the spike are depicted red and in sticks in one subunit only. Bottom: Side view of pb4 spike. C termini are indicated (C). (C) Superimposition of pb4 spike in isosurface view of the tip (green) and Tip-FhuA (blue) maps (unsharpened). Three slices are shown, and their position along the spike is indicated in (B). The map after interaction with the receptor contains the spike decoration domains and the FNIIIs while that before interaction contains only pb4 spike. Inset: pb4 spike decoration domains and linkers are colored in different shades of blue on one pb4 subunit [linker 1 (l1, residues 484 to 547), domain 1 (d1, 548 to 566), linker 2 (l2, 548 to 566), domain 2 (d2, 567 to 618), and linker 3 (l3, 519 to 626)].

Fig. 5.. Proposed mechanism of trigger for infection.

(A) Scheme of T5 tail tip. The hdIV-FNIII linker (black) and the plug (brown) are highlighted in BHP_pb3, as well as the FNIII-spike linker (blue), loop 224-232 of the third FNIII (salmon), and the orientation of the proximal three β strands of the spike (black arrows) in pb4. (B) Following RBP_pb5-FhuA interaction, a constraint (1) would induce a twisting of the proximal pb4 spike (2), pulling on pb4 FNIII-spike linker. This in turn would destabilize the FNIII string network (3). (C) Blow up on pb4 FNIII-spike interface before (left) and after (right) interaction with FhuA. The two spikes are aligned on the middle sheet of the spike (residues 413 to 465). pb4 subunits are in different shades of green, the FNIII-spike linker in blue, and FNIII loop 224-232 in salmon. (D) The FNIII string reorganization around pb4 spike induces pb4 bending, brings the tube close to the membrane, and disengages BHP_pb3 hdIV-FNIII linker (4). This latter event liberates the plug, opening the tube (5) and destabilizing TMP_pb2C, which is expelled (6). (E) BHP_pb3 plugs refold as a β-hairpin legs and anchor in the outer membrane (OM), TMP_pb2* is also expelled, its C termini inserting in the crevice between BHP_pb3 subunits, its hydrophobic segment inserting in the OM to form a channel. TMP_pb2C, released in the periplasm, would digest the peptidoglycan (PG). In (E), colored boxes depict proteins that could be modeled (full line) or for which densities are visible (dotted line). TMP_pb2C, for which no densities is visible but for which we propose a location, is represented as an empty Pacman.