Mechanism Of LuxS/AI-2 Type Quorum-sensing System Regulated By LsrR On Intracellular Survival Of Avian Pathogenic Escherichia Coli

APEC（Avian Pathogenic Escherichia coli）has complex serotypes,many virulence factors and increasing drug resistance,which has brought serious economic losses to the poultry industry.The virulence of APEC is regulated by many factors,among which the quorum sensing system（Quorum sensing,QS）can regulate a variety of biological characteristics,such as bacterial virulence,bioluminescence,biofilm formation,drug resistance and so on,by sensing the concentration of self-inducer（Autoinducer,AI）.LsrR is a transcriptional regulator of the Escherichia coli LuxS/AI-2 quorum sensing system,which regulates the transcription of the lsroperon（lsrRK,lsrACDBFG）gene and then regulates the internalization,phosphorylation and degradation of signal molecules AI-2.It has been reported that LsrR regulates the oxidative stress response of Escherichia coli.Hydrogen sulfide（H₂S）,as the second messenger,participates in the protection of bacteria against oxidative damage induced by reactive oxygen species（ROS）and antibiotics.Previous studies have shown that lsrR deletion can significantly enhance the antioxidant stress ability and survival ability of APEC in macrophages,but the molecular mechanism is not clear.It has been reported that LsrR can regulate the oxidative stress response of Escherichia coli.Hydrogen sulfide（H₂S）,as the second messenger,is involved in the protection of bacteria against oxidative damage induced by reactive oxygen species（ROS）and antibiotics.It is not clear whether LsrR regulates APEC antioxidant stress through sulfur metabolism.Therefore,this study analyzes the regulation of sulfur metabolism by LsrR and then regulates the antioxidant stress of APEC,and affects the interaction between APEC and macrophages,which provides a theoretical basis for the prevention and control of APEC.The specific research contents are as follows:1 LsrR is involved in regulating the biological characteristics of sulfur metabolism and the biofilm of APECIn this study,lsroperon gene related deletion strains E940ΔlsrB,E940ΔlsrF,E940ΔlsrK,E940ΔlsrR,E940ΔlsrRΔlsrB,E940ΔlsrRΔlsrF,E940ΔlsrRΔlsrK were constructed by CRISPR-Cas9 system.The extracellular AI-2 internalization of the above deleted strains was detected by signal molecular detection technique.the results showed that the extracellular AI-2 internalization of E940ΔlsrB,E940ΔlsrRΔlsrK decreased;the extracellular AI-2internalization of E940ΔlsrR and E940ΔlsrRΔlsrF strains accelerated;E940ΔlsrF,E940ΔlsrK strains had no effect on extracellular AI-2 internalization.The above results show that lsrB and lsrR affect the process of AI-2 internalization.The effects of lsrB,lsrF,lsrK and lsrR deletions on the biological characteristics of APEC were investigated from the point of view of biofilm formation,oxidative stress and H₂S production,respectively.the results showed that lsrB,lsrF and lsrK deletions had no significant effect on the biofilm forming ability of E940,while lsrR deletion significantly enhanced the biofilm forming ability of E940.After lsrR deletion,the antioxidant stress ability of E940 was significantly increased by about 3 times.H₂S,as the second messenger,had antioxidant stress.The results showed that the deletion of lsrB,lsrF and lsrK had no significant effect on the production of H₂S in E940 cells,but the deletion of lsrR significantly increased the production of H₂S in E940 cells.H₂S is involved in antioxidant stress,but the mechanism of lsrR regulating sulfur metabolism is still unclear.2 LsrR regulates sulfur metabolism in APEC by binding to the promoter region of genes related to sulfur metabolismPrevious studies have shown that lsrR deletion leads to the increase of H₂S in E940 cells and the enhancement of antioxidant stress ability,but the mechanism is unclear.In order to analyze its mechanism from the perspective of etiology,transcriptome sequencing of lsrR deleted and wild strains was carried out.Transcriptome data analysis showed that after lsrR deletion,SO₄^2-transport gene cysPUWA,cysteine transport related gene cyu P and cysteine synthesis gene cysND,cysC,cysI,cysH,transcriptional level were up-regulated about 3times.In order to further explore the effect of LsrR on the transcriptional level of the above genes,the transcriptional level of sulfur metabolism related genes after overexpression of lsrR was verified by constructing overexpression plasmid pBAD33-lsrR.The results showed that the transcriptional level of sulfate transport gene cysD was down-regulated about 3 times after overexpression of lsrR.The activity of the cysD promoter was verified byβ-galactosidase,but the unbinding of LsrR and cysD promoter regions was verified by EMSA.The regulation of lsrR on sulfur metabolism needs further investigation.3 Study on the regulatory pathway of LsrR on RAW264.7 in mouse macrophagesPrevious studies have shown that lsrR deletion can significantly enhance the viability of APEC in macrophages.In order to explore the effect of lsrR on the interaction between APEC and macrophages through sulfur metabolism,the adhesion and invasion ability of E940 and E940ΔlsrR were detected.The results showed that lsrR deletion significantly enhanced the adhesion and invasion ability of E940 to macrophage RAW264.7.The results of flow cytometry showed that compared with wild strains,the content of ROS and apoptosis in RAW264.7 increased significantly after E940ΔlsrR infection,indicating that lsrR was involved in the interaction between APEC and macrophages.In order to further explore the regulation pathway and hydrogen sulfide synthesis of macrophages infected with lsrR deletion APEC,the transcription levels of related cytokines were detected by fluorescent quantitative PCR.The results showed that compared with wild strains,E940ΔlsrR infected macrophages RAW264.7 mainly caused the transcription levels of TLR4,My D88 and NF-KB to be up-regulated about 1-3 times,and the transcription levels of IL-12 and CCL2 in innate immune response pathway were up-regulated about 1-2 times.The transcriptional levels of acquired immune cytokines IL-6 and IFN-γwere up-regulated by 1-3 times.There was no significant change in the transcription level of sulfur metabolism gene CSE and apoptosis related genes Caspase-1,Caspase-3 and Caspase-9.These results suggest that lsrR participates in the intracellular survival and ROS production of macrophages activated by APEC.RAW264.7 in macrophages infected with lsrR deletion APEC binds to TRL4receptors and activates NF-κB pathway,but has no effect on intracellular hydrogen sulfide synthesis and apoptosis transcription..In summary,by exploring the effects of transcriptional regulators lsrR on the biological characteristics of APEC,it was proved that the deletion of lsrR promoted the internalization of extracellular AI-2,significantly enhanced the ability of APEC biofilm formation,antioxidant stress and intracellular H₂S production.By exploring the regulatory pathway of LsrR on mouse macrophages,it was proved that E940 infected RAW264.7 increased intracellular survival,intracellular reactive oxygen species production and apoptosis after lsrR deletion.It was further proved that macrophages infected with lsrR deletion APEC promoted the up-regulation of NF-κB pathway gene transcription by binding to TRL4 receptor.