SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly
SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly
复制标题
DOI：
10.1111/cmi.13154
复制
发表时间：
2020-01-11
影响因子：
3.4
通讯作者：
Sansom, Fiona M.
中科院分区：
文献类型：
--
作者：
Bitew, Mebratu A.;Hofmann, Janine;Sansom, Fiona M.
伯内特柯克斯体是人畜共患疾病 Q 热的病原体，是一种革兰氏阴性细菌，在巨噬细胞内高度氧化的液泡内复制。转座子突变体文库的筛选表明，编码假定的短链脱氢酶的 sdrA 是细胞内复制所必需的。短链脱氢酶是 NADP(H) 依赖性氧化还原酶，SdrA 含有预测的 NADP(+) 结合位点，表明它可能促进 C. burnetii 的 NADP(H) 再生，这是生存氧化应激的关键过程。纯化的重组 6xHis-SdrA 能够在体外将 NADP(+) 转化为 NADP(H)。预测的 NADP 结合位点内第 12 位保守甘氨酸残基突变为丙氨酸，消除了显着的酶活性。 sdrA 突变体 (sdrA::Tn) 与质粒表达的 SdrA 的互补将细胞内复制恢复到野生型水平，但表达酶促无活性的 G12A_SdrA 却没有。 sdrA::Tn 突变体在体外对氧化应激更敏感，用抗氧化剂 L-抗坏血酸处理受感染的宿主细胞，部分挽救了 sdrA::Tn 的细胞内生长缺陷。最后，稳定同位素标记研究表明，sdrA::Tn 中代谢途径的通量变化与氧化应激增加的存在一致，并且与 C. burnetii NMII 相比，感染 sdrA::Tn 的宿主细胞的活性氧水平升高。
Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP(+) binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6xHis-SdrA was able to convert NADP(+) to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.