Structure of defense against restriction proteins DarA and Hdf in phage P1 reveals a new molecular mechanism during phage assembly, infection and DNA ejection

TL;DR

This study reveals how phage P1 uses DarA and Hdf proteins to protect its DNA and assemble its structure, offering insights into phage defense mechanisms.

Contribution

The study provides the first structural details of DarA-Hdf complexes in phage P1 and their role in DNA protection and capsid assembly.

Findings

DarA and Hdf form 55 pairs at penton-hexon junctions in the phage head, interacting with DNA.

These complexes are absent in the contracted P1 without DNA, suggesting a role in DNA protection and assembly.

The complexes have a hydrophilic outer surface, possibly aiding solubility in the host cytoplasm.

Abstract

The continuous “arms race” between bacterial antiviral defense systems and phage anti-defense strategies drives evolutionary innovation. Previous study indicated that the defense against restriction (Dar) proteins DarA and Hdf in myophage P1 are associated with the head morphogenesis. However, the structural information for these proteins was lacking, and the mechanisms by which they mediate head morphogenesis and protect phage DNA against bacterial defense systems remained poorly understood. Using cryo-electron microscopy (cryo-EM), we resolved the entire structures of extended P1 and contracted P1 with partial DNA, with the latter lacking the baseplate, as well as the head structure of contracted P1 without DNA. We identified the structural proteins for the P1, including the head, connector complex, and baseplate, which exhibited conserved properties among the majority of myophages with a simple baseplate. Notably, 55 DarA-Hdf pairs are attached to the inner surface of head at each penton-hexon junction in the extended P1 and contracted P1 with partial DNA. The DarA and Hdf together form a complex that is tightly bound to the capsid and interacts with the DNA. However, these pairs are absent in the contracted P1 without DNA. Based on our three states of P1, we hypothesis that these extensive interactions among DarA, Hdf, DNA, and head play crucial roles in mediating capsid assembly, enhancing capsid stability, and protecting phage DNA. Our results provide a structural basis for further exploration of the mechanism by which Dar proteins function during phage assembly, infection and DNA ejection. This molecular mechanism may be conserved among P1-like phages.

Bacteria have evolved a range of defense mechanisms, such as restriction-modification (R-M) systems, to destroy invading phage DNA, posing a major challenge to phage therapy. However, phages have evolved counter-defenses. Notably, myophage P1 encodes defense against restriction (Dar) proteins, including DarA and Hdf, to protect its genome. However, the structural basis and protection mechanism for these Dar proteins remain to be elucidated. In this study, we focused on the head structures of the bacteriophage P1 in its extended state and two types of contracted state. We discovered that DarA and Hdf proteins form 55 pair complexes that are anchored at each penton-hexon junction on the inner surface of the head in the extended P1 and the contracted P1 with partial DNA. The complex has a positively charged inner surface, thus interacting with the DNA. Additionally, the complex has a hydrophilic outer surface that maybe necessary to maintain the solubility of the DNA-protein complex in the host cytoplasm following injection. However, these 55 pairs are absent in the contracted P1 without DNA. This study unveils the molecular details of intricate interactions among DarA, Hdf, DNA, and capsid in different states, thereby uncovering a sophisticated phage defense mechanism and providing structural insights for engineering phages to better overcome bacterial resistance.

Figures