Structure and function of a C. difficile bacteriophage

The C. difficile cell is coated with a crystalline protein array known as the S-layer, the structure of which was recently solved. As very few phages that infect S-layer-containing Gram-positive bacteria have been studied, it is not known how phages interact with and penetrate the S-layer as well as the thick cell wall and subsequent cell membrane. Consequently, our work focuses on understanding the 3D structural molecular detail of phages to try to elucidate their binding and penetration mechanics of this multi-layered Gram-positive cell envelope. To achieve this, we have determined the structure of a C. difficile myophage using Cryo-EM and single particle analysis.

All tailed bacteriophages are made up of several connecting structures starting at the capsid which contains the phage genome. Within a specific pentameric vertex of the icosahedral capsid, a portal complex attaches to the rest of the phage, and acts as the exit for the genome; the portal connects to the tail via a neck structure; the neck then interacts with the tail structure which feeds into the baseplate and needle structures. Determining the molecular structures of these phage components could highlight key targets for genetic engineering. φ-MMP03 was successfully propagated and purified enabling the determination of a whole virion structure. These structures were built de novo into experimentally determined cryo-EM density maps ranging from 3 Å to 3.9 Å resolution.

The whole structure of φ-MMP03 comprised 13 fittable proteins. Interestingly, the portal adaptor and tail adaptor lacked accurate homologues. The needle tip architecture of φ-MMP03 was novel, lacking several domains thought to be conserved amongst all phages which may hint at a Gram-positive-specific needle tip architecture. In addition, a dual enzymatic domain within the phage baseplate was discovered which demonstrated peptidoglycan-degrading activity. This enzymatic activity highlights a multi-step phage infection process that differs from previously characterised C. difficile infecting phage.