Extended Data Fig. 3: Validation of tagged constructs for defense activity, Mhp co-purification, NAD⁺ cyclization, and chemical standards.
From: Bacterial TIR-based immune systems sense phage capsids to initiate defense
(a) Tenfold serial dilutions of Φ80a-vir phage on lawns of S. aureus RN4220 harboring plasmids carrying either untagged or hexahystidyl- or Flag-tagged versions of ThsB1 or ThsB2, along with untagged ThsA. (b) LC-MS/MS identification of ~35 kDa protein isolated with ThsB1 and ThsB2 from infected S. aureus RN4220 cells shown in Fig. 3b. Gel slices (n = 3) were subjected to reduction, alkylation, and in-gel digestion. Peptides were extracted before being injected for LC-MS/MS analysis. Search results against the staphylococcal phage ΦNM1 proteome are presented as the percentage of sequence coverage (ΣCoverage) versus the indication of unique identified peptides (ΣPSMs). Data for the phage Mhp is shown in red. (c) Cyclization reaction of oxidized nicotinamide adenine dinucleotide (NAD + ) mediated by the ThsB1/B2/Mhp complex, which yields 1”-3’- glyocyclic ADP-ribose (1”-3’-gcADPR) and nicotinamide (NAM). (d) HPLC analysis of different commercially available chemicals used in this study.
