The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae
The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae
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Na-转位 NADH：醌氧化还原酶增强霍乱弧菌细胞质中的氧化应激
DOI：
10.1128/jb.00342-16
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发表时间：
2016
影响因子：
3.2
通讯作者：
J. Steuber
中科院分区：
文献类型：
--
作者：
V. Muras;Paul Dogaru;Yusuke Minato;C. Häse;J. Steuber
摘要：我们在人类病原体霍乱弧菌的细胞质中寻找活性氧（ROS）的来源，并利用染料2 ',7 ' -二氯荧光素（DCFH-DA）在呼吸细胞中研究ROS的形成机制。通过比较有无活性Na+易位NADH：醌氧化还原酶（Na+-NQR）的霍乱弧菌菌株，确定了这种呼吸性钠离子氧化还原泵是体内ROS的产生者。在产生Na+-NQR变异的霍乱弧菌细胞中检测到的细胞质ROS的数量与相应膜组分形成超氧化物的速率密切相关。与缺乏Na+-NQR的突变体膜（0.18±0.01 μmol min - 1 mg - 1）相比，野生型霍乱弧菌膜的超氧化物生成活性（9.8±0.6 μmol超氧化物min - 1 mg - 1）有所增加。在nqr缺失菌株中，过表达质粒编码的Na+-NQR导致超氧化物的形成急剧增加（42.6±2.8 μmol min−1 mg−1）。通过分析一种缺乏醌还原活性的Na+-NQR变体，我们确定了胞质NqrF亚基的还原黄素腺嘌呤二核苷酸（FAD）辅因子是霍乱弧菌胞内超氧化物形成的位点。讨论了Na+-NQR形成超氧化物对霍乱弧菌毒力的影响。几项研究表明，霍乱弧菌中的Na+-NQR以一种未知的方式影响毒力。我们确定了Na+-NQR的nadh氧化NqrF亚基中减少的FAD辅因子是霍乱弧菌细胞质中超氧化物形成的位点。我们的研究提供了一个框架来理解在呼吸过程中形成的活性氧如何参与从有氧到嗜微氧（肠道）栖息地过渡过程中毒力因子的调控表达。这一假设可能对许多其他病原体是正确的，比如霍乱弧菌，它们依赖Na+-NQR作为唯一的电致NADH脱氢酶。
ABSTRACT We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na+-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min−1 mg−1 membrane protein) compared to membranes from the mutant lacking Na+-NQR (0.18 ± 0.01 μmol min−1 mg−1). Overexpression of plasmid-encoded Na+-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min−1 mg−1). By analyzing a variant of Na+-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na+-NQR on the virulence of V. cholerae is discussed. IMPORTANCE In several studies, it was demonstrated that the Na+-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na+-NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na+-NQR as the sole electrogenic NADH dehydrogenase.
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Na( )-转位 NADH:醌氧化还原酶在霍乱弧菌电压产生和 Na( ) 挤出中的作用
DOI：
10.1016/j.bbabio.2015.12.010
发表时间：
2016
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Vorburger;Nedielkov;Brosig;Schunke;Steffen;Möller;Steuber
通讯作者：
Steuber
影响因子：
4.3
作者：
Ohnishi, S. Tsuyoshi;Shinzawa-Itoh, Kyoko;Ohnishi, Tomoko
通讯作者：
Ohnishi, Tomoko
影响因子：
7.4
作者：
Marchesi, E;Rota, C;Mason, RP
通讯作者：
Mason, RP
影响因子：
2.9
作者：
Kundu TK;Velayutham M;Zweier JL
通讯作者：
Zweier JL
影响因子：
2.8
作者：
Minato, Yusuke;Fassio, Sara R.;Haese, Claudia C.
通讯作者：
Haese, Claudia C.