Phage-encoded NARP3 system rebuilds NAD⁺ to subvert bacterial immunity.

Abstract

Phages deploy diverse countermeasures to evade bacterial nicotinamide adenine dinucleotide (NAD⁺)-directed immunity in a perpetual arms race. Known pathways include NAD⁺ reconstitution pathway 1 (NARP1), which recycles adenosine 5'-diphosphate-ribose, and NARP2, which converts nicotinamide into NAD⁺ via nicotinamide mononucleotide ligation. Whether additional NAD⁺-restoring strategies exist has remained unclear. Here, we identify NARP3, a conserved two-gene NAD⁺-restoring phage pathway widespread in Enterobacteriaceae-infecting phages. NARP3 encodes a pyridine nucleoside uptake system C (PnuC)-like nicotinamide riboside transporter (Bas30_87) and a bifunctional NAD⁺ biosynthesis regulator (NadR)-like enzyme (Bas30_86). Bas30_87 imports nicotinamide riboside, and Bas30_86 converts it to NAD⁺ through sequential phosphorylation and adenylation. NARP3 fully restores NAD⁺ pools depleted by Sir2-HerA defenses, enabling robust phage replication. We solve x-ray crystal structures of Bas30_86 alone and bound to NAD⁺, revealing coordinated substrate capture, intermediate handling, and product formation. Mutational analyses confirm that both transport and enzymatic activities are essential. NARP3 functions as a metabolite-centered countermeasure, expands the phage arsenal, and underscores NAD⁺ metabolism as a central battlefield in host-phage conflicts. Its discovery provides a blueprint for engineering phages to bypass NAD⁺-dependent bacterial immunity and offers a mechanistic framework to harness metabolite-guided viral strategies for biotechnological applications.