Systematic identification and validation of cyclic nucleotide receptors in bacteria

Abstract

Cyclic nucleotide-based signaling plays a central role in bacterial antiviral defense systems such as CBASS. These systems rely on a diverse array of cyclic nucleotide second messengers (cNSMs) to initiate immune responses upon phage infection. However, the full spectrum of protein receptors that detect these molecules remains largely uncharacterized. This study aims to expand our understanding of cNSM sig-naling, specifically involving cyclic UMP, cyclic UMP-AMP, cyclic GMP-UMP, and cyclic AMP-AMP-GMP, by identifying novel receptor proteins in bacteria and experimentally validating their interactions. ☐ To achieve this, proteomic mass spectrometry analyses were performed on pull-down assays using bacterial cell lysates, with the cNSMs as baits. Candidate receptors were identified based on selective enrichment and further prioritized using bioinformatic analysis. Subsequent biochemical validation involved gene cloning, recombinant protein expression, and purification. Differential scanning fluorimetry (DSF) was employed as a primary screening technique to assess ligand-induced stabilization of protein targets. ☐ Our findings demonstrate that this systematic workflow, combining pull-down proteomics with DSF screening, offers a robust, efficient, and reliable methodology for receptor identification. Importantly, we identified a promising candidate protein that binds cyclic UMP (cUMP), highlighting a potentially novel receptor involved in cNSM-mediated signaling. These results could potentially contribute to our understanding of bacterial signaling networks and regarding the immune regulation mechanisms.