Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Abstract

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3–10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3′3′-cyclic GMP–AMP define a mechanism of metal-independent hydrolysis 3′ of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.

Genetic mining of newly isolated Salmophages for phage therapy

“…One has to keep in mind that small ORFs in bacteriophages can encode small proteins enabling phages to evade host anti-phage systems. Among them are found: anti-CRISPR proteins (Acr) such as ACR3112-12 (52 aa) from Pseudomonas phage D3112 [52]; proteins preventing superinfection such as the immunity protein Imm (83 aa) of bacteriophage T4 [53] or the lipoprotein Llp (77aa) of bacteriophage T5 [54]; anti-RM proteins such as Ocr (117 aa) of bacteriophage T7 [55]; the newly discovered anti-CBASS Acb (about 94 aa) found in an increasing number of phages of various phylogenetic background such as Pseudomonas phages PaMx33, 35, 41 and 43 as well as the enterobacteriophage T4 [19, 20].…”

“…Undoubtedly, co-evolution works both ways, and phages therefore evolved their own arsenal to counter cellular defenses. One can cite anti-RM or anti-CRISPR proteins [18] as well as the newly discovered anti-CBASS and anti-Pycsar proteins [19, 20]. Given the diversity of anti-phage defenses systems, a matching diversity of yet unknown anti-cellular defenses functions is probably buried into the phage genomic “dark-matter” awaiting discovery.…”

Turning down the (C)BASS: Phage-encoded inhibitors jam bacterial immune signaling

“…Where there is defense, there is counter-defense. A paper by Hobbs et al (2022) reconfirms that this emerging tenet of the arms race between bacteria and their predators also holds true for phage defense systems that rely on secondary messenger signals.…”

Bacteriophage anti-defense genes that neutralize TIR and STING immune responses

“…Cyclic nucleotide messengers generated by these enzymes mobilize cognate death effectors, including nucleases, proteases, pore-forming toxins, as well as sirtuin and Toll-interleukin-1 receptor (TIR) domain NAD-cleaving enzymes (Kazlauskiene et al, 2017; Lowey et al, 2020; Ofir et al, 2021), through direct binding to an effector-linked cyclic nucleotide sensor, such as the widespread STING (stimulator of interferon genes) domain. Many viruses, in turn, deploy specialized nucleases that rapidly cleave and inactivate cyclic nucleotides, extinguishing host immune responses (Athukoralage et al, 2020; Hobbs et al, 2022). Other virus-encoded proteins act as molecular ‘sponges’ that sequester nucleotide immune messengers (Leavitt et al, 2022).…”

“…Cyclic nucleotide messengers generated by these enzymes mobilize cognate death effectors, including nucleases, proteases, pore-forming toxins, as well as sirtuin and Toll-interleukin-1 receptor (TIR) domain NAD-cleaving enzymes (11)(12)(13), through direct binding to an effector-linked cyclic nucleotide sensor, such as the widespread STING (stimulator of interferon genes) domain. Many viruses, in turn, deploy specialized nucleases that rapidly cleave and inactivate cyclic nucleotides, extinguishing host immune responses (14,15). Currently, our understanding of how viruses evade the multitude of known Abi systems is limited, and little is known about whether viruses employ counter-defense strategies beyond signal degradation.…”