NAD synthesis is required for intracellular replication of , the causative agent of the neglected zoonotic disease Q fever.

is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that , encoding a putative l-aspartate oxidase required for NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the mutant compared with the WT, confirming the involvement of NadB in NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of NadB, and expression of WT and R275L GST-NadB fusion proteins in JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between NAD synthesis and intracellular replication of This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat infections.