Phage protease enzymes activate CBASS antiphage immunity

Area of Science:

• Microbiology
• Molecular Biology
• Immunology

Background:

• Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are crucial bacterial defense mechanisms against phage infection.
• These systems rely on cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes to detect invaders and trigger antiviral responses.
• The diversity of CBASS operons highlights their significant role in prokaryotic immunity.

Purpose of the Study:

• To investigate the mechanism of CD-NTase activation in bacterial antiviral defense.
• To identify how CBASS systems recognize and respond to specific phage activities.
• To elucidate the role of phage proteases in triggering CBASS-mediated immunity.

Main Methods:

• In vitro reconstitution of CBASS recognition of phage T4 infection.
• Biochemical assays to analyze enzyme activity and cleavage.
• Structure-based phylogenetic analyses of CD-NTase enzymes and activation loops.

Main Results:

• Proteolytic cleavage by phage prohead proteases was identified as a key mechanism for activating CD-NTase enzymes.
• Cleavage of a surface-exposed activation loop triggers enzyme catalysis and the synthesis of immune signaling nucleotides.
• Phage prohead proteases were shown to be sufficient for activating CBASS in vivo, indicating their role in late-stage infection defense.
• Specific residues controlling CBASS recognition of different phage families were mapped.

Conclusions:

• CBASS systems function as molecular sensors of phage protease activity, representing a widespread antiviral defense strategy.
• The interaction between phage proteases and bacterial CD-NTase enzymes is a critical determinant of bacterial immunity.
• Understanding this mechanism provides insights into the co-evolution of bacteria and phages and potential targets for novel antimicrobials.