Screening And Identification Of The Genes Regulated By SEN2967 And SEN3610 In Salmonella Enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis(S.Enteritidis,SE)is a gram-negative enteropathogen,which is highly pathogenic to humans and animals.Transcription factors are considered as the proteins that bind to specific nucleotide sequences on specific genes and regulate the transcription of these genes.Transcription factors can regulate the expression of virulence factors and participate in the process of physiological and biochemical metabolism.Therefore,the study of transcription factors in Salmonella show great significance for the research of pathogenic mechanism,prevention and control of Salmonella.In the previous study of our lab,the SCOTS technology was used to determine and screen genes expressed by SE-infected chicken macrophages.We found SEN2967 and SEN3610 genes were up-regulated significantly during the infection process compared with cultivation in vitro.But there has been no report about the functions of both regulators in Salmonella.In this study,we have successfully constructed the gene deleted strains which were named as C50041?SEN2967 and C50041?SEN3610 by homologous recombination system,and used two-dimensional electrophoresis(2D)and iTraQ to analyze the proteins regulated by both regulators.Construction and identification of the biological characteristics of the deleted mutant strains and inducible expression strains of S.EnteritidisqRT-PCR analysis was performed to compare the transcription level of mRNA for both regulators between cell infection and cultivation in vitro conditions.The expression of SEN2967 was up-regulated by about 98 times,and SEN3610 was up-regulated by about 15 times at 5 h post-infection.In this study,we have successfully constructed the deleted mutant strains C50041?SEN2967,C50041?SEN3610,C50041?SEN2967*pBad24,C50041?SEN3610*pBad24 by ?-Red recombination system and inducible expression strains C50041?SEN2967(pBad24-SEN29673*Flag),C50041?SEN3610(pBad24-SEN36103*Flag).The results of Western Blot showed that the SEN2967 and SEN3610 protein was successfully expressed in inducible expression strain.Among the three strains C50041,C50041?SEN2967 and C50041?SEN3610,there were no obviously differences in growth curve and biochemical properties.Competition experiment in vivo showed that both the pathogenicity of C50041?SEN2967 and C50041?SEN3610 were significantly lower than C50041.The successful construction of gene deletion strains for both regulators and inducible expression strains provided the corresponding biological materials for the study of proteins regulated by SEN2967 and SEN3610.Proteomic Study of genes regulated by SEN2967 and SEN3610 in S.EnteritidisIn this study,the differences in the protein expression profile of SEN2967 and SEN3610 deletion strains and their corresponding inducible expression strains were studied by 2D technique.Firstly,extract the whole bacterial protein of the strains respectively,and ensure the same loading quantity of sample.Two-dimensional electrophoresis was then performed and the 2D map was obtained by silver staining,followed with analysis using PD Quest software.There were 56 differentially expressed protein spots between C50041 ?SEN2967*pBad24 and C50041?SEN2967(pBad24-SEN29673*Flag),while 91 differentially expressed protein spots were detected betweeen C50041 ?SEN3610*pBad24 and C50041?SEN3610(pBad24-SEN36103*Flag).The results of iTraQ showed that according to the criteria for defining differentially expressed proteins(fold change ratio?1.5 and p?0.05),there were 81 up-regulated proteins and 90 down-regulated proteins between C50041?SEN2967*pBad24 and C50041?SEN2967(pBad24-SEN29673*Flag),25 up-regulated proteins and 21 down-regulated proteins between C50041?SEN2967*pBad24 and C50041?SEN2967(pBad24-SEN29673*Flag).These results demonstrated that deletion of SEN2967 and SEN3610 could cause changes in bacterial protein expression profile.In the results of iTraQ,proteins associated with T3SS were found in the down-regulated proteins of C50041?SEN2967*pBad24 and C50041?SEN3610*pBad24,including the effector proteins of SPI-1 and SPI-5,as well as the proteins associated with the composition,assembly and length of the Needle Complex(NC)structure.qRT-PCR analysis confirmed that the genes probably regualted by SEN3610 was higher expressed in inducible expression strain than deleted strain.In this study,the proteins regulated by SEN2967 and SEN3610 laid the foundation for the further study of transcriptional regulation of SE involved in the signal pathway.Validation of genes regulated by SEN2967 and SEN3610 using EMSAIn this study,the prokaryotic expression bacteria for SEN3610 was successfully constructed by pMAL-5 vector.We obtained a series of recombinant strains,including E.coli ER2523(pMAL-c5X-SEN3610)and E.coli ER2523(pMAL-p5X-SEN3610).Approximately 40 mg of MBP-SEN3610 fusion protein was purified and dialyzed for EMSA experiments.According to the sequences of identified genes by iTRAQ,the promoter sequence was amplified to be decorated as the 5'-FAM fluorescently labeled probes by two-stage PCR.The binding ability between MBP-SEN3610 and each probe was detected by EMSA reaction system.The results showed that SEN3610 had obvious binding ability to the promoter of hilA,hilD and pipD.We speculated that SEN3610 could regulate expression of genes belonging to T3SS through HilA and HilD regulators.