Three-dimensional structure of tropism-switching Bordetella bacteriophage

Abstract

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail "hub," six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7A resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.

Fig. 1.

CryoET of the BPP-1 particles. (A) A representative tomography series with tilt range of −60° to 60°. (B) The top-to-bottom sectional views of a BPP-1 particle cut from the tomogram reconstructed from the tilt series shown in A. The thickness of each section is 7.86 Å, and the distance between two sections is 104 Å. Yellow arrow, tail spikes; red arrow, distal end of the tail fibers. (C) Averaged tail fiber. (D) Shaded surface representation of the composite BPP-1 map combining the averaged map of tail fibers (C) with that of the head and tail after averaging of 60 particles. The 11 pentons are colored in pink. The tail assembly sits on top of one vertex. (E) Side view of the BPP-1 structure showing the relative distances (in Å) between the tail, spikes, and the tail fibers.

Fig. 2.

Localization of mtd to the distal ends of the tail fibers by 3D reconstruction of immunogold-labeled particles. (A and B) Images of immunogold-labeled BPP-1 particles. The phage particles are incubated with rat anti-mtd antibody, followed by incubation with anti-rat, 5-nm gold-conjugated secondary antibodies. The gold particles are shown specifically attached to the tail fibers with very clean background (average <1 gold per μm²). (C) Section from a 3D electron tomogram reconstructed from the particle shown in B, demonstrating the location of mtd. The arrow points to the two gold particles that are localized to the distal end densities of the same tail fiber. (D) Fitting of the crystal structure of the Mtd trimer (PDB ID code 1YU4) to the tail fiber distal end densities. Two Mtd trimers are modeled to the bottom portion of the distal end densities. Each trimer fits into one lobe with the variable region facing down.

Fig. 3.

Subnanometer resolution reconstruction of the head revealed the fold of the MCP and the accessory stabilizing or cementing proteins. (A) A focal pair of cryoEM images of the BPP-1 particles embedded in vitreous ice. (Upper) The close-to-focus image that has high resolution information of the particles. (Lower) The far-from-focus image, which has high contrast for orientation determination. (B) Shaded surface view of the head. Three 6-fold axes and one 3-fold axis in the front are labeled. The dashed line box represents two MCP and two cementing protein from two neighboring hexons. (C) Fold of the MCP. One MCP monomer is extracted and fitted to the crystal structure of the HK97 MCP gp5. A long α-helix was identified at the P domain. Two short helices and some β-sheets are also visible and fit well with similar secondary structure elements present in the gp5 atomic structure. Domains indicated include axial (A) domain, peripheral (P) domain, and elongated (E) loop. (D) The interaction between MCP and cementing protein. The cementing protein is a elongated globular density that localized at the 2-fold axis as a dimer. The cementing proteins interact with the E domain and end of P domain of its adjacent MCPs to provide extra forces for the integrity of the capsid. In HK97, capsid stability is achieved through a completely different mechanism involving chemical bonds formed by the E loop. Blue, MCP; red: cementing protein.

Fig. 4.

Improved Mtd avidity during BPP-1 attachment to host cell. (A) A proposed model of attachment. (1) When a phage particle contacts a host bacteria, Mtd molecules on one of the six tail fiber ends first reaches the cell membrane receptor, retaining the particle near the cell surface. (2) Cooperative attachment. The initial binding of Mtd molecules on one tail fiber increases the probability of random collisions leading to the binding of other Mtd molecules on the same particle, thus providing a phage particle an increased strength of attachment to a cell surface. (3) Binding of multiple tail fibers to the cellular membrane receptors orients the phage particles toward the cell surface. Lateral movement of the Mtd-bound receptors may pull the particle toward the cell surface, providing possible mechanical support for the tail insertion. Because the height difference between the tail and the surrounding tail spikes is smaller than the average thickness of the periplasm, the tail spikes have to undergo a conformational change to alleviate the space hindrance. (4) Penetration of the tail through the periplasm to the inner membrane triggers the release of DNA into the cell. The attachment of tail spikes to the capsid shell suggest that conformational changes of the tail spikes during tail insertion might be the signal passed to the portal complex via the capsid shell to trigger DNA release. (B) Thin section EM image of BPP-1 infecting Bordetella bacteria, showing BPP-1 attachment spread uniformly across the bacterial surface.