The CueR-regulated transporters CopA and CusFABC coordinate copper detoxification in Vibrio parahaemolyticus

Vibrio parahaemolyticus 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 V. parahaemolyticus. Through RNA sequencing, we show that most genes in the CueR and Fur regulons are significantly upregulated during V. parahaemolyticus response to external Cu. Quantitative real-time PCR (qRT-PCR) showed that copA and cusA are strongly induced by Cu. Growth curve analyses and spot dilution assay revealed that CopA and CusFABC protect V. parahaemolyticus 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 V. parahaemolyticus resistance to pyrithione. In a competitive-infection assay, the copA and cusFABC 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 copA and cusFABC, and positively regulate their expression. Collectively, V. parahaemolyticus utilizes the CueR-regulated transporters CopA and CusFABC to coordinate Cu detoxification and colonization of the zebrafish intestine.