Journal of Biological Chemistry
Volume 293, Issue 48, 30 November 2018, Pages 18636-18645
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Microbiology
De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever

https://doi.org/10.1074/jbc.RA118.005190Get rights and content
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Coxiella burnetii 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 C. burnetii 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 nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo 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 Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli 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 C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii. This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.

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This work was supported in part by Australian National Health and Medical Research Council Grants APP1063646 and APP1120344 (to H. J. N.). The authors declare that they have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S4.

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The abbreviations used are:

    CCV

    Coxiella-containing vacuole

    RT

    retention time

    NaMN

    nicotinic acid mononucleotide

    GST

    glutathione S-transferase

    OAA

    oxaloacetate

    TCA

    tricarboxylic acid

    NMII

    Nine Mile phase II

    DMEM

    Dulbecco's modified Eagle's medium

    FCS

    fetal calf serum

    qPCR

    quantitative PCR

    DAPI

    4′,6′-diamino-2-phenylindole.