Molecular Mechanism Of Inhibition Of The Bacterial CBASS Immune System By Phage Proteins

One of the immune response strategies that widely exist in organisms is sensing the signal molecules produced or carried by the virus to trigger the downstream immune response.In mammals,viral double-stranded DNA(ds DNA)binding to cyclic GMP-AMP synthase(cGAS)in the cytoplasm activates cGAS activity to catalyze the formation of 2’,3’-c GAMP,which acts as a signaling molecule that binds to downstream STING effector proteins and induces type I interferon responses.Recently,thousands of cGAS-like enzymes named cGAS/Dnc V-like nucleotidtransferases(CD-NTase)have also been identified in bacteria.These enzymes are activated during phage infection by unknown mechanisms and produce cyclic oligonucleotides that act as second messengers to activate downstream effector proteins.This bacterial immunization strategy has been defined as the Cyclic-oligonucleotide Based Anti-phage Signaling System(CBASS).When faced with bacterial defense mechanisms,phages have developed various resistance strategies.In this thesis,we investigated the mechanism of Acb2(anti-CBASS 2),a protein of the phage anti-bacterial CBASS system.First,we conducted in vitro biochemical experiments that did not detect any interaction between Acb2 and the key proteins of CBASS system.However,we found that Acb2 could efficiently bind to the second messenger 3’,3’-c GAMP of CBASS system without exhibiting enzymatic activity during this binding process.Based on the binding relationship between Acb2 and 3’,3’-c GAMP,we analyzed the structure of Acb2 protein and its complex binding to 3’,3’-c GAMP.The structure showed that Acb2 protein behaves as a compact hexamer.Each of the two N-termini of Acb2 monomer binds one 3’,3’-c GAMP molecule,and the hexamer binds a total of three 3’,3’-c GAMP molecules.By adsorbing and sequestering 3’,3’-c GAMP,Acb2 effectively abrogated the immune function of CBASS system.Based on Acb2 binding to 3’,3’-c GAMP,this thesis further demonstrated that Acb2 interacts with a variety of cyclic oligonucleotides,including c-di-AMP,3’,3’-c-di-UMP,3’,3’-c UA,3’,3’-c UG,3’,2’-c GAMP and eukaryotic second messenger 2’,3’-c GAMP.This indicates that Acb2 can act as a broad-spectrum inhibitor of the CBASS system.The structure of Acb2 complex with c-di-AMP was further elucidated and c-di-AMP was found to bind Acb2 in a similar manner as 3’,3’-c GAMP.In summary,this thesis reveals the molecular mechanism by which Acb2 acts as a “sponge protein” to antagonize the broad spectrum of bacterial CBASS immune system,proposes a novel strategy for phage to suppress the CBASS system immunity,and expands our understanding of viral evasion strategy based on cGAS-like bacterial immune system.