RNA Chaperone Hfq Regulates The Virulence And Drug Resistance Of Shigella Sonnei

ObjectiveTo analyze the regulation of hfq gene on the virulence and drug-resistant phenotype of Shigella sonnei,understand its regulatory mechanism,discover new sRNAs those can regulate the virulence and drug resistance of Shigella sonnei,which provides new ideas and drug targets for the prevention and treatment of Shigella infectious diseases.Methods1.The λ-Red homologous recombination method was performed to construct hfq gene knockout strain of Shigella sonnei.2.HeLa cell invasion test and mouse competitive lung invasion test were used to detect differences of virulence phenotypes between Shigella sonnei wild strain and hfq gene knockout strain.3.The minimum inhibitory concentration(MIC)of Shigella sonnei wild strain and hfq gene knockout strain was determined by micro-broth dilution method.The antibiotics for detection included cefuroxime,ceftriaxone,cefotaxime,kanamycin,gentamicin,amikacin,chloramphenicol,aztreonam,sulfamethoxazole,imipenem,polymyxin B,mecillinam,meropenem,and ampicillin.4.Transcriptome sequencing was performed on Shigella sonnei wild strain and hfq gene knockout strain.Rockhopper software was used to predict sRNA of Shigella sonnei,and screen out the differential sRNAs.RNAhybrid and RIsearch software were used to predict the target mRNAs of differential sRNAs.The sRNA related to the virulence and drug resistance of Shigella sonnei was selected by analyzing the function of the target gene.5.Extract the RNA of Shigella sonnei wild strain and hfq gene knockout strain,and differentiate genes such as ompC,ompF,gadA,gadB,hdeA,hdeB,PhoP,sRNA1043,sRNA8,sRNA9,sNA1203,sRNA372,sRNA841,sRNA330 and sRNA912 were verified by qPCR verification.Results1.hfq gene knockout strain of Shigella sonnei was successfully constructed:WT/Δhfq.2.In the HeLa cell invasion experiment showed that WT/Ahfq:kan was weaker invasiveness to HeLa cells than WT,and the invasion index was less than 1,P<0.001;the mouse competitive lung invasion experiment showed that the ability of invasive to the lung of Babl/C mice of WT/Ahfq:kan was weaker than that of WT,and the invasion index was less than 1,P<0.001.The deletion of the hfq gene leads to the expression level of the virulence factor ipah9.8,acid-resistance genes gadA,gadB,hdeA、hdeB,and two-component system sensor histidine kinase envZ,two-component signal transduction system PhoP/PhoQ were changed,which regulated the virulence of Shigella sonnei.3.The minimum inhibitory concentration of WT/Δhfq for some antibiotics had been reduced,including cefuroxime,ceftriaxone,cefotaxime,kanamycin,gentamicin,amikacin,chloramphenicol,aztreonam,and sulfamethoxazole,the minimum inhibitory concentration for polymyxin B,mecillinam,meropenem,imipenem and ampicillin have no change.The deletion of hfq gene led to the changes in expression of TolC gene and outer membrane pore protein OmpC and OmpF,which regulated the drug resistance of Shigella sonnei.4.The prediction sRNAs related to the drug resistance of Shigella sonnei in this study included sRNA1043,sRNA1065,sRNA8,sRNA9,sNA1203,sRNA1128,sRNA372,sRNA841,and sRNA912,their target genes involved in the formation of biofilms,cell membrane composition and permeability.5.The prediction sRNAs related to the virulence of Shigella sonnei in this study included sRNA1043,sRNA1065,and sRNA330,their target genes involved in regulating the composition of bacterial endotoxins and pathogenicity.Conclusions1.The RNA chaperone Hfq can regulate the resistance and virulence of Shigella sonnei:after hfq gene is knocked out,the sensitivity to antibiotics such as cephalosporin,aminoglycoside,chloramphenicol,aztreonam,and sulfamethoxazole increased,and there is no change in the sensitivity of carbapenems,polymyxin B and other antibiotics.Both the HeLa cell invasion experiment and the mouse competitive lung invasion experiment showe that the deletion of hfq gene significantly reduced the virulence of Shigella sonnei.2.The hfq gene can regulate the virulence of Shigella sonnei by regulating the transcription levels of virulence factors,acid-resistance genes,and two-component systems;and regulate drug resistance by regulating the transcription levels of efflux pump system and outer membrane proteins.3.The prediction sRNAs related to the drug resistance of Shigella sonnei in this study included sRNA1065,sRNA 8,sRNA9,sNA1203,sRNA1128,sRNA372,sRNA841,and sRNA912.The prediction sRNAs related to the virulence of Shigella sonnei in this study included sRNA1043,sRNA 1065,and sRNA330.