Pangenomics facilitated with structural analysis reveals host NAD+ manipulation as a major virulence activity of bacterial effectors

Nicotinamide adenine dinucleotide (NAD+) has emerged as a major component in both eukaryotic and prokaryotic immune systems. Recent findings that Toll/interleukin-1 receptor (TIR) proteins function as NAD+ hydrolases (NADase) link NAD+ metabolism with immune signaling. In this study, we investigated how manipulation of host NAD+ metabolism can be employed as a virulence strategy by pathogens. Using the pangenome of the model bacterial pathogen Pseudomonas syringae, we conducted a structure-based similarity search for type III effectors (T3Es) with potential NADase activity from 35,000 orthogroups. Thirteen T3Es, including five novel ones, were identified to possess domain(s) related to seven NAD+-hydrolyzing enzyme families. Most P. syringae strains encode at least one NAD+-manipulating T3Es, and many have multiple. We experimentally confirmed the type III-dependent secretion and virulence activity of a novel T3E, which shows similarity with both TIR and adenosine diphosphate ribose (ADPR) cyclase. Using AI-based structural prediction and in silico molecular docking, we found divergence at the NAD+-interacting positions and confirmed the functional importance of these residues in the TIR T3E HopAM1. This study highlights NAD+ manipulation as a key virulence mechanism and provides insights into how pathogens may promote infection through manipulation of host NAD+ metabolism.