Phage protease enzymes activate CBASS antiphage immunity

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are immunity pathways in bacteria that use a cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme to sense phage infection and initiate antiviral defense. Bacteria encode thousands of diverse CBASS operons, demonstrating a critical role for CD-NTase activation in controlling the prokaryotic response to viral infection. Here we discover proteolytic cleavage by phage prohead proteases as a mechanism of CD-NTase activation and demonstrate that diverse CBASS operons function as molecular sensors of protease activity. We reconstitute CBASS recognition of phage T4 infection in vitro and identify proteolytic cleavage of a surface exposed CD-NTase activation loop as a trigger of enzyme catalysis and nucleotide immune signal synthesis. Phage prohead proteases are sufficient to activate CBASS in vivo and explain how immune signaling is initiated during late stages of viral infection. Combining biochemical and structure-based phylogenetic analyses, we map activation loops in Clade A, D, and G CD-NTase enzymes and define critical residues that control CBASS recognition of distinct phage families. Our results define CBASS recognition of phage protease activity as a widespread mechanism of antiviral defense.