Structural features of E. coli Stx bacteriophage phi24B revealed with cryo-electron microscopy

This study utilizes high-resolution cryo-electron microscopy and proteomics to reveal the detailed structural architecture of the Shiga toxin-converting bacteriophage phi24B, characterizing its T=9 icosahedral capsid, complex tail assembly, and unique peripheral features that distinguish it from related podoviruses.

The Gist

The Big Picture: A Tiny Trojan Horse

Imagine a microscopic "Trojan Horse" that doesn't carry soldiers, but carries a deadly toxin. This is the phi24B bacteriophage. It is a virus that infects E. coli bacteria.

Usually, E. coli is harmless (or even helpful) in our guts. But when phi24B infects it, the bacteria turns into a factory that produces Shiga toxin, a poison that can cause severe kidney failure in humans. This paper is like a high-resolution X-ray and a parts list that finally reveals exactly what this "Trojan Horse" looks like and how it works.

1. The Body: A Giant, Ornate Ball

Most viruses are simple spheres. Phi24B is a bit more complex.

• The Capsid (The Shell): Think of the virus's head as a soccer ball made of 20 triangles. But this isn't a standard soccer ball; it's a giant, intricate geodesic dome (about 74 nanometers wide).
• The "Studs" (Decorating Proteins): The surface of this ball isn't smooth. It's covered in tiny, hexagonal "studs" (proteins called gp84).
  • The Twist: These studs are actually scissors. They are enzymes that can cut through mucus (the slimy layer in our intestines).
  • The Surprise: When the virus is first made inside the bacteria, these scissors are full-sized. But as soon as the virus pops out of the bacteria, it "snaps" itself. It cuts off the heavy, useless handle, leaving only the sharp, functional tip attached to the shell. This allows the virus to swim through the mucus of the gut to find new bacteria.

2. The Tail: The Injection Needle

At the bottom of the ball is a short, sturdy tail. It looks like a complex mechanical plug.

• The Portal: This is the door where the DNA lives. It's a twelve-sided ring that acts as a gatekeeper.
• The Nozzle: Below the door is a hexagonal nozzle.
• The Needle: Sticking out of the nozzle is a long, flexible, spring-like needle (the gp56 protein).
  • The Analogy: Imagine a dart gun. The virus is the gun, the tail is the barrel, and the needle is the dart.
  • The Mystery: The paper suggests that this needle is the key. It likely touches the bacteria first. Once it locks onto the right door (a protein on the bacteria called BamA), the needle might snap off or change shape, triggering the release of the virus's genetic payload.

3. The "Ejection Protein": The Giant Rope

Inside the virus, there is a massive protein called gp47.

• The Size: It is huge—over 2,800 amino acids long.
• The Function: The scientists couldn't see it in their 3D models because it's hidden inside. They think it acts like a giant rope or a tunnel.
• The Metaphor: When the virus hits the bacteria, it doesn't just shoot DNA out like a bullet. It likely shoots out this giant rope first. This rope drills through the bacterial cell wall, creating a tunnel so the DNA can slide through safely to the inside of the bacteria.

4. Two States: "Loaded" vs. "Empty"

The researchers took pictures of the virus in two different states:

1. Intact (Loaded): The virus is full of DNA. The tail is slightly tilted, and the internal "rope" (gp47) is packed tight.
2. Ejected (Empty): The virus has fired its DNA. The internal structure changes slightly, the DNA is gone, and the "rope" is missing.

• Why it matters: By comparing the two, they saw that the virus is a rigid machine that doesn't fall apart when it fires; it just empties its cargo.

5. Why This Matters

• The "Big Six" Problem: This virus is related to the "Big Six" dangerous strains of E. coli that cause food poisoning outbreaks.
• The Mucus Trick: The fact that the virus has these "mucus-cutting scissors" (gp84) explains how it survives in the human gut, which is a slimy, hostile environment. It can chew through the mucus barrier to find its prey.
• Evolutionary Mystery: The virus looks very similar to a virus that infects Pseudomonas bacteria (a different type of germ), even though they are very different genetically. This suggests that nature has found a "perfect design" for a virus shell and tail, and it keeps reusing that blueprint, just swapping out the "weapons" (the fibers) to target different bacteria.

Summary

This paper is like a forensic investigation of a microscopic assassin.

• The Weapon: A virus that turns bacteria into toxin factories.
• The Armor: A giant, icosahedral shell covered in mucus-cutting studs.
• The Trigger: A flexible needle that likely unlocks the bacteria.
• The Delivery: A giant internal rope that builds a tunnel for the genetic code.

By understanding exactly how this machine is built, scientists hope to figure out how to stop it, potentially leading to new ways to treat dangerous infections caused by Shiga-toxin-producing E. coli.

Technical Summary

1. Problem Statement

Shiga toxin-converting bacteriophages (Stx-phages) are critical drivers in the emergence of pathogenic Escherichia coli (STEC) strains, which cause severe foodborne illnesses and Hemolytic Uremic Syndrome (HUS). While the genetic organization of these phages (specifically the lambdoid group) is well-studied, detailed structural information regarding their virion architecture, particularly for the highly prevalent phi24B phage, has been scarce.

• Knowledge Gaps: It was unknown which specific proteins interact with the host receptor (BamA), how the phage penetrates the O-antigen barrier, and the structural organization of its unique genome (57.7 kb, larger than typical lambdoid phages).
• Technical Challenge: Previous attempts to characterize phi24B structurally failed due to difficulties in culturing and purifying high-concentration stocks suitable for cryo-electron microscopy (cryo-EM).

2. Methodology

The authors employed a multi-disciplinary approach combining virology, proteomics, and high-resolution structural biology:

• Sample Preparation: Phi24B was induced from a lysogenic E. coli 4sR strain using Mitomycin C. Virions were purified via sucrose gradient ultracentrifugation and Freon cushioning to achieve high titers ($6.5 \times 10^{12}$ PFU/mL).
• Proteomics:
  • Gel-based: SDS-PAGE followed by in-gel trypsin digestion and LC-MS/MS.
  • Solution-based: Batch trypsinolysis of the whole virion for quantitative protein abundance estimation.
  • Analysis: Used MaxQuant and Andromeda to identify structural proteins and estimate copy numbers relative to the portal protein.
• Cryo-Electron Microscopy (Cryo-EM):
  • Data collected on a Titan Krios (300 kV) with a Gatan K3 camera.
  • Dataset: ~88,000 particles were analyzed, distinguishing between intact virions (containing DNA) and ejected virions (DNA released).
  • Processing: Single-particle analysis using CryoSPARC v4.7.1. Both icosahedral (C1) and asymmetric reconstructions were performed. Local refinements were applied to the capsid, tail, and portal regions to achieve atomic resolution.
• Negative Stain TEM: Used to visualize flexible regions (specifically the C-terminal tip of the tail needle) that were unresolved in cryo-EM due to flexibility.
• Modeling: De novo atomic models were built using AlphaFold2 predictions fitted into cryo-EM density maps, followed by real-space refinement in Phenix and ISOLDE.

3. Key Contributions & Results

A. Overall Virion Architecture

• Capsid: The virion features an isometric icosahedral capsid (~74 nm diameter) with T=9 symmetry, a rare trait among podoviruses (typically T=7). This larger capsid accommodates the large 57.7 kb genome.
• Tail: A short tail (~22 nm) comprising a dodecameric portal, two rings of adapter proteins, a hexameric nozzle, six lateral fibers, and a central needle.
• Conformational States: The study resolved two distinct states:
  1. Intact: DNA packed in four concentric layers; tail is tilted ~2° relative to the capsid axis.
  2. Ejected: DNA released; a single DNA layer remains bound to the inner capsid. The tail aligns with the central axis, suggesting internal DNA pressure influences tail orientation.

B. Capsid Composition and Proteins

• Major Capsid Protein (MCP, gp69): Adopts an HK97-like fold but lacks the covalent "chainmail" isopeptide bonds found in many HK97-like phages, relying instead on non-covalent electrostatic interfaces.
• Cementing Protein (gp67): Assembles into trimers at the three-capsomer junctions, forming a "tulip-like" structure that stabilizes the capsid.
• Decorating Protein (gp84): A processed esterase protein (related to E. coli NanS) that decorates the capsid.
  • Processing: The full-length protein (68 kDa) undergoes autoproteolytic cleavage after virion release. The C-terminal catalytic domain and a central domain are shed, leaving only the N-terminal DUF1737 domain (35 kDa) attached to the capsid.
  • Location: Forms a hexamer at the center of the icosahedral faces (central hexons).
  • Function: Proposed to facilitate mucin binding or degradation, aiding in environmental persistence and transmission.

C. Tail Assembly and Receptor Binding

• Portal Complex (gp72): A dodecamer connecting the capsid to the tail. The barrel domain interacts with the DNA.
• Adapters: Two concentric dodecameric rings (gp64 and gp62) connect the portal to the nozzle.
• Nozzle (gp57): A hexamer capping the tail.
• Lateral Fibers (gp61): Six trimers attached to the nozzle. The N-terminal domain binds the tail, while the C-terminal domain is highly flexible (collagen-like shaft) and likely does not mediate the primary receptor binding.
• Central Needle (gp56): A flexible trimeric fiber protruding from the nozzle.
  • Mechanism: The N-terminal domain is anchored in the nozzle. Upon receptor binding, the needle likely detaches, triggering DNA ejection.
  • Receptor Interaction: The distal tip of the needle (C-terminal $\beta$-prism) is flexible and unresolved in cryo-EM but shows an additional density in negative stain TEM. This density likely corresponds to gp54/gp55, which are hypothesized to be the primary Receptor Binding Proteins (RBPs) interacting with the host's BamA protein.

D. Ejection Protein (gp47)

• A massive protein (~2811 aa) identified via proteomics but absent from the external structure. It is proposed to be packaged inside the capsid as an ejection protein, potentially forming a transperiplasmic conduit to degrade the peptidoglycan layer during infection.

4. Significance

• Structural Conservation vs. Divergence: The phi24B virion core (capsid and tail) is structurally homologous to the Ralstonia phage GP4, despite low sequence identity. This suggests a conserved structural module (T=9 capsid + specific tail architecture) that has been adapted for different hosts.
• Mechanism of Infection: The study clarifies the infection mechanism, proposing that the flexible central needle (potentially with gp54/55) mediates BamA recognition, while the lateral fibers (gp61) may play a secondary or structural role.
• Lysogenic Conversion & Virulence: The discovery of the processed gp84 esterase on the virion surface provides a structural basis for how Stx-phages might interact with the gut mucin layer, potentially explaining their ability to spread and convert commensal E. coli into pathogenic STEC strains even in the presence of O-antigens.
• Therapeutic Implications: Understanding the specific receptor interactions and structural vulnerabilities (e.g., the unique T=9 symmetry and specific fiber arrangements) could inform the development of phage therapy or anti-virulence strategies against STEC outbreaks.

5. Conclusion

This paper provides the first high-resolution structural map of the phi24B bacteriophage, revealing a complex virion with a T=9 capsid, a unique proteolytically processed capsid-decorating esterase, and a specialized tail assembly. The findings bridge the gap between the genetic potential of Stx-phages and their physical mechanisms of host recognition and infection, offering new insights into the evolution of bacterial pathogenicity.