Investigating the Process of Sheath Maturation in Antifeeding Prophage: a Phage Tail-Like Protein Translocation...

Area of Science:

• Microbiology
• Structural Biology
• Molecular Biology

Background:

• The antifeeding prophage (Afp) from Serratia entomophila is a model external contractile injection system (eCIS).
• Unlike contractile phages and pyocins, Afp utilizes three sheath proteins instead of one.
• Understanding the assembly of these unique sheath proteins is crucial for eCIS function.

Purpose of the Study:

• To investigate the distinct roles of Afp2, Afp3, and Afp4 sheath proteins in Afp assembly.
• To elucidate the interactions between Afp2, Afp3, and Afp16 (TrP) during sheath maturation.
• To develop a model for Afp sheath assembly and maturation.

Main Methods:

• Targeted mutagenesis of Afp sheath protein genes (afp2, afp3, afp4).
• Transmission electron microscopy (TEM) to visualize mutant assembly defects.
• Pulldown assays to study protein-protein interactions (Afp2, Afp3, Afp4, Afp16).
• In vitro complementation assays to restore biological activity.

Main Results:

• Afp2 and Afp3 form alternate hexameric stacks constituting the helical sheath.
• Afp3 interacts with Afp16 to regulate sheath length; Afp2 is vital for sheath polymerization and baseplate assembly.
• Mutations in afp2, afp3, or afp4 resulted in distinct assembly defects, including incomplete sheath formation and lack of tail fibers.
• Afp3 forms an SDS-resistant complex with Afp2, indicating a direct interaction.

Conclusions:

• Afp2 and Afp3, despite sequence similarity, have distinct and essential roles in Afp assembly.
• Afp3's interaction with Afp16 is key for controlling sheath and tube length.
• Afp2 is critical for sheath polymerization onto the baseplate.
• The proposed model provides insights into eCIS assembly, guiding the use of Afp and related systems as nanodelivery vehicles.