Mechanism Of The Copper Ion Tolerance And Type Ⅲ Secretion System Controlled By Transcriptional Regulator CorR In Edwardsiella Piscicida

Edwardsiella piscicida is a Gram-negative pathogen that can infect numerous aquaculture fish and cause serious economic losses.Copper and iron elements play important roles in life activities,regulating each other’s metabolism and mediating important pathogen-host interactions.Copper ions have significant bactericidal effects and participate in the host innate immune response against pathogen infections;bacteria also have evolved to tolerate the toxic effects of copper ions over long periods of time.Therefore,a deep understanding of bacterial mechanisms for the copper ion tolerance is of great significance for the development of novel antibacterial agents or measures.In this study,a defined mutant library was employed for transposon insertion sequencing（TIS）analysis to systematically dissect genes supplemented with 0.5 and 3 mmol/L copper ions,and 269 copper ion tolerance-related genes were obtained,including ETAE₂324（thereafter named corR）and copA.Further research showed that CopA is a copper ion efflux pump of E.piscicida,and its deletion significantly reduced bacterial tolerance to copper ion;CorR is a transcriptional regulator that can increase the expression of copA to enhance the tolerance of E.piscicida to copper ions,thus validating our TIS analyses.The analysis of the possible biological pathways involved in these 269 genes showed that they mainly participate in the repair process of bacterial membrane and DNA damage after copper intoxication,the regulation process of maintaining copper stability,and the transport and metabolism of nutrients,indicating that copper ions have a broad impact on bacterial growth and metabolism in the bacterium.The virulence factors type Ⅲ secretion system（T3SS）and type Ⅵ secretion system（T6SS）play important roles in the colonization of E.piscicida in host cells.We found that CorR enhanced eseB transcript levels in response to copper ions,thereby promoting the secretion of the T3SS structural component EseB.Competition index（CI）assays revealed that the deletion of CorR decreased the infectious lethality of E.piscicida against turbot.Meanwhile,CorR and CopA promoted the internalization and replication of E.piscicida in HeLa and J774A.1 cells.These results illustrated that CorR not only coordinates tolerance to copper ions but also is involved in the regulation of E.piscicida virulence system.In addition,the regulatory gene iscR was previously identified as the conditional essential gene in E.piscicida related to the bacterial in vivo colonization in fish.The gene iscR encodes a transcriptional regulator of the iscRSUA-hscBA-fdx operon related to iron-sulfur cluster synthesis.IscR inhibited the transcription of eseB,thereby decreasing EseB secretion.However,according to the results of CI assays,the deletion of iscR reduced the colonization of E.piscicida in turbot,further validating that iscR is an in vivo conditional essential gene.We speculated that IscR deletion may allow E.piscicida to prematurely activate eseB expression during infection,leading to recognition and clearance by the host immune system,and ultimately reduced the infectious lethality of E.piscicida against turbot.In conclusion,the explorations of CorR,CopA,and IscR facilitate a better understanding of the mechanisms of virulence regulation in E.piscicida,and lay a solid foundation for future studies on the reciprocal regulation between copper tolerance and iron-sulfur cluster.It also provides new ideas for the development of efficient fish attenuated vaccines and novel antibacterial strategies.