| Object:On the basis of food isolates of Listeria monocytogenes Lm928,the genes of Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326 in LIPI-4 were deleted,and the complement strains were constructed for verification.The effects of these three genes on the biological characteristics of Listeria monocytogenes,such as sugar fermentation,growth curve,biofilm formation,regulation of other virulence factors at transcription level,bacterial adhesion and invasion and intracellular proliferation,and median lethal dose(LD50)in mouse models were compared and analyzed,so as to provide reference for further research on the pathogenic mechanism and prevention and control of Listeria monocytogenes.It laid the foundation for screening the major genes of Lm928 LIPI-4.Methods:（1）The deletion mutants of Lm928ΔLm4b＿02324,Lm928ΔLm4b＿02325 and Lm928ΔLm4b＿02326 were constructed by SOE-PCR method.The chloramphenicol resistance positive strains were screened and identified by PCR at 42℃.Three gene deletion strains with stable inheritance were successfully constructed by successive generations.（2）We amplified CLm4b＿02324,CLm4b＿02325 and CLm4b＿02326 gene callback fragments.They were connected to the shuttles and electrocuted into the deletion strains to obtain CLm928ΔLm4b＿02324,CLm928ΔLm4b＿02325 and CLm928ΔLm4b＿02326 gene complement strains.At30℃,kanamycin resistance positive strains were screened and identified by PCR and double digestion.（2）（1）The effects of Lm928 on sugar fermentation were compared with each gene deletion strain and its complement strain;（2）the growth curves of Lm928,each gene deletion strain and its complement strain were measured under different temperature,Na Cl,p H and ethanol conditions.（3）The biofilm formation ability of Lm928 and the gene deletion strains and their complement strains was determined.（3）（1）Lm928 and Lm928ΔLm4b＿02325 were cultured in BHI medium,and the expressions of other LIPI-4 genes were detected by Real-time PCR.（2）The expression of other virulence factors of Listeria monocytogenes was detected by Real-time PCR after Lm928 was cultured with BHI medium and the gene deletion strains and their complement strains.（3）HBMECs were infected with MOI 10 and cultured for 12h.Bacterial RNA was extracted and the expressions of other virulence factors of Listeria monocytogenes were detected by Real-time PCR.（4）（1）LD50 was determined after Lm928 was infected with each gene deletion strain and its complement strain.（2）Analysis of the adhesion,invasion and intracellular proliferation of human microvascular endothelial cells induced by Lm928,gene deletion strains and their complement strains.Results:（1）Three stable genetic deletion strains were successfully constructed:Lm928ΔLm4b＿02324,Lm928ΔLm4b＿02325 and Lm928ΔLm4b＿02326.Three gene complement strains CLm928ΔLm4b＿02324,CLm928ΔLm4b＿02325 and CLm928ΔLm4b＿02326 were constructed successfully.（2）Sugar fermentation test showed that the deletion of Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326 genes had no effect on Lm928 sugar fermentation.The deletion of Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326genes did not change the tolerance of bacteria under acid-base,salt and ethanol environment.Deletion of Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326 genes had no significant effect on biofilm formation.（3）Real-time PCR was used to detect the expression of other LIPI-4 genes in Lm928ΔLm4b＿02325.The results showed that the expression of other genes decreased significantly except Lm4b＿02324.In BHI culture,Real-time PCR detected that the virulence factors of each gene deletion strain were mostly down-regulated in Lm928ΔLm4b＿02324 and Lm928ΔLm4b＿02325,while the Plc B and Inl B of Lm928ΔLm4b＿02326 were significantly up-regulated.During intracellular proliferation of HBMECs,Real-time PCR detected the expression levels of virulence factors of each deletion strain,and the virulence factors of Lm928ΔLm4b＿02324 and Lm928ΔLm4b＿02325 showed a downward trend,while the hly and Inl C of Lm928ΔLm4b＿02326 were significantly up-regulated.（4）The ability of Lm928ΔLm4b＿02324,Lm928ΔLm4b＿02325 and Lm928ΔLm4b＿02326 to HBMEC adhesion and proliferation was stronger than that of Lm928.Lm4b＿02324 and Lm4b＿02325 gene deletion enhanced the virulence of Lm928,while the deletion of Lm4b＿02326 gene had no significant effect on the virulence.Conclusion:In this study,the deletion strains and corresponding complement strains of Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326 genes were successfully constructed on the basis of 4b serotype food strain Lm928.The results showed that Lm4b＿02324,Lm4b＿02325 and Lm4b＿02326 genes did not play a regulatory role in sugar fermentation,resistance to environmental stress and biofilm formation.The deletion of the Bgl G protein encoded by the Lm4b＿02325 gene can terminate the transcription of the downstream gene of LIPI-4.In the intracellular proliferation culture of BHI and HBMEC,Lm4b＿02324 and Lm4b＿02325 positively regulated the expression of other important virulence genes of Listeria monocytogenes,while Lm4b＿02326 showed negative regulatory characteristics.The deletion of Lm4b＿02324 and Lm4b＿02325 genes enhanced the virulence of LM at the level of infection in animal models,while the deletion of Lm4b＿02324,Lm4b＿02325and Lm4b＿02326 genes enhanced the virulence of LM at the level of infection in cultured cells. |
