De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever
De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever
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DOI：
10.1074/jbc.ra118.005190
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发表时间：
2018-11-30
影响因子：
4.8
通讯作者：
Sansom, Fiona M.
中科院分区：
文献类型：
--
作者：
Bitew, Mebratu A.;Khoo, Chen Ai;Sansom, Fiona M.
贝氏柯克斯体（Coxiellaburnetii）是一种胞内革兰氏阴性细菌，可引起重要的人畜共患Q热。改进的遗传工具和在宿主无细胞培养基中培养这种细菌的能力推进了对C。Burnetii的发病机制，但机制，使其能够生存的敌意吞噬溶酶体内仍然不完全了解。以前筛选的转座子突变体库在HeLa细胞内复制表明，nadB，编码一个假定的L-天冬氨酸氧化酶从头NAD合成所需的，是需要细胞内复制。在这里，使用两个独立的nadB突变体和细胞内复制试验的遗传互补，我们证实了这一发现。非靶向代谢物分析表明，与WT相比，nadB突变体中NAD生物合成途径中的代谢物发生了关键变化，证实了NadB参与了NAD的从头合成。生物信息学分析显示在275位存在功能保守的精氨酸残基。利用定点突变技术将此残基替换为亮氨酸，使大肠杆菌NadB活性丧失，并在大肠杆菌中表达了WT和R275 L GST-NadB融合蛋白。coli JM 109中，我们发现纯化的重组WT GST-NadB具有L-天冬氨酸氧化酶活性，而R275 L NadB变体则无活性。C.用表达该失活R275 L NadB的质粒构建的贝氏疟原虫nadB突变体不能将复制恢复到WT水平，证实了从头NAD合成和胞内复制之间的联系。伯内特氏菌这表明，靶向这种原核特异性途径可以促进治疗C。贝氏体感染
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.