CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and...

Area of Science:

• Microbiology
• Immunology
• Structural Biology

Background:

• Cyclic GMP-AMP synthase/DncV-like nucleotidyltransferase (CD-NTase) enzymes are crucial for innate immunity in bacteria and animals, synthesizing second messenger nucleotides to trigger defense responses.
• Bacterial CD-NTases are involved in various cellular processes, including antiviral defense, but the receptors that sense their signals remain largely uncharacterized.

Purpose of the Study:

• To identify and characterize novel bacterial receptors that respond to CD-NTase-synthesized nucleotide second messengers.
• To elucidate the structure and mechanism of action of these newly discovered receptors.

Main Methods:

• Structural biology techniques (X-ray crystallography) to determine the high-resolution structures of Cap4.
• Biochemical assays to investigate the ligand-binding properties and enzymatic activity of Cap4 and related proteins.
• Bioinformatic analysis to identify the prevalence and diversity of Cap4-like proteins across bacterial species.

Main Results:

• Discovery of Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a representative of a large family (>2,000 members) of bacterial CD-NTase signal receptors.
• Structural determination of Cap4 revealing a DNA endonuclease domain and an appended SAVED domain, a fusion of two CRISPR-associated Rossman fold (CARF) subunits.
• Demonstration that the SAVED domain specifically recognizes and discriminates between different linkage isomers (2'-5' and 3'-5') of cyclic oligonucleotide second messengers, enabling the detection of at least 180 distinct species.

Conclusions:

• SAVED CARF family proteins are identified as major receptors for nucleotide second messengers in bacterial cyclic nucleotide-based adaptive systems (CBASS) and CRISPR-Cas immune systems.
• The findings highlight the critical role of linkage specificity in receptor-ligand interactions, extending this principle beyond mammalian cGAS-STING signaling.
• This work expands our understanding of bacterial innate immunity and the diversity of nucleotide second messenger signaling pathways.