The CueR-regulated transporters CopA and CusFABC coordinate copper detoxification in <i>Vibrio parahaemolyticus</i>

<i>Vibrio parahaemolyticus</i> is a zoonotic pathogen that poses a serious threat to shrimp aquaculture and public health worldwide. Copper (Cu) serves as a catalytic cofactor and structural element in bacteria but is toxic in excess. Herein, we report that the CueR-regulated transporters CopA and CusFABC coordinate Cu detoxification in <i>V. parahaemolyticus</i>. Through RNA sequencing, we show that most genes in the CueR and Fur regulons are significantly upregulated during <i>V. parahaemolyticus</i> response to external Cu. Quantitative real-time PCR (qRT-PCR) showed that <i>copA</i> and <i>cusA</i> are strongly induced by Cu. Growth curve analyses and spot dilution assay revealed that CopA and CusFABC protect <i>V. parahaemolyticus</i> against Cu-induced bactericidal effects. Inductively coupled plasma-mass spectrometry revealed that CopA and CusFABC are involved in maintaining Cu and Fe homoeostasis. Additionally, CopA promotes <i>V. parahaemolyticus</i> resistance to pyrithione. In a competitive-infection assay, the <i>copA</i> and <i>cusFABC</i> double mutant, but not the single mutants, was significantly attenuated in colonization of the zebrafish intestine. Lastly, qRT-PCR, RNA sequencing, β-galactosidase activity, and AIDmut-Seq analyses revealed that CueR can bind to the promoters of <i>copA</i> and <i>cusFABC</i>, and positively regulate their expression. Collectively, <i>V. parahaemolyticus</i> utilizes the CueR-regulated transporters CopA and CusFABC to coordinate Cu detoxification and colonization of the zebrafish intestine.副溶血性弧菌（Vibrio parahaemolyticus）是一种人畜共患病原体，对全球虾类养殖业与公共健康均构成严重威胁。铜（Cu）作为细菌体内的催化辅因子与结构组成元件，过量时则会产生毒性。本研究发现，受CueR调控的转运蛋白CopA与CusFABC可在副溶血性弧菌中协同介导铜解毒过程。通过RNA测序，我们证实副溶血性弧菌在应对外界铜胁迫时，CueR与Fur调节子中的绝大多数基因均出现显著上调。实时荧光定量聚合酶链反应（qRT-PCR）结果显示，copA与cusA基因可被铜强烈诱导表达。生长曲线分析与斑点稀释试验表明，CopA与CusFABC可保护副溶血性弧菌免受铜诱导的杀菌作用。电感耦合等离子体质谱法分析显示，CopA与CusFABC参与维持铜与铁的稳态平衡。此外，CopA可提升副溶血性弧菌对吡啶硫酮的抗性。在竞争性感染试验中，copA与cusFABC双突变体（而非单突变体）在斑马鱼肠道内的定殖能力显著减弱。最后，结合RNA测序、实时荧光定量聚合酶链反应、β-半乳糖苷酶活性检测与AIDmut-Seq分析，我们证实CueR可结合copA与cusFABC的启动子区域，并正向调控其表达。综上，副溶血性弧菌通过CueR调控的CopA与CusFABC转运蛋白协同完成铜解毒过程，并促进其在斑马鱼肠道内的定殖。