Effectors of cell death in bacterial antiphage defense

Abstract

Bacteria encode many systems to detect and respond to infection with bacteriophages. Cyclic-oligonucleotide based antiphage signaling systems (CBASS) are widespread antiviral systems encoded in approximately 10% of bacterial genomes. Upon infection of CBASS-encoding bacteria, nucleotide second messengers are synthesized and can diffuse throughout the cell to bind diverse effector proteins. We use structural and biochemical methods to characterize effector proteins that specifically recognize nucleotide second messengers and are activated to induce cell death. Once this abortive infection system is activated, the effector proteins kill the bacterial host before the phage is able to replicate, thereby halting phage infection. We characterize one family of effectors, Cap4 proteins, that use a SAVED domain to specifically recognize nucleotide second messengers and are then activated to kill the cell through the indiscriminate cleavage of double-stranded DNA. These data highlight SAVED domains as widespread ligand-binding domains found in many CBASS effectors and revealed an evolutionary connection between CBASS and CRISPR immunity. We also characterized effector proteins that contain transmembrane domains, revealing that they target the inner membrane to induce cell death after phage infection. These data highlight membrane disruption as a widespread strategy to induce cell death in CBASS immunity. We further characterize one family of transmembrane effectors, Cap15 proteins, which we show use a minimal β-barrel domain to recognize nucleotide second messengers. Together, these studies begin to characterize diverse CBASS effectors that fulfill two requirements: 1. specific recognition of nucleotide second messengers, either by SAVED or β-barrel domains and 2. the induction of cell death, either through destruction of nucleic acids by Cap4 nucleases or inner membrane disruption by Cap15 transmembrane effectors. These data support an emerging model where CBASS effectors use a modular domain architecture to sense second messengers and induce cell death to halt phage replication.