Construction And Biological Characterization Of GreAgreB Gene Deletion Strain Of Salmonella Enteritidis

Salmonella Enteritidis can infect a variety of livestock and poultry,and infect humans through meat,eggs,milk and other products,causing human food poisoning.It is an important zoonotic and foodborne pathogen.In order to investigate the prevalence of Salmonella in large-scale chicken farms in Handan and its surrounding areas,a multiplex PCR method was established using the genomes of Salmonella Enteritidis（ATCC13076）,Salmonella Typhimurium（ATCC14028）,Salmonella Pullorum（ATCC13036）and Salmonella Gallinarum（ATCC9184）as templates.A total of 343 strains of Salmonella were isolated by multiplex PCR from 6500 chicken farm samples,and the isolation rate of salmonella was 5.28%.Among them,295 Salmonella Enteritidis strains,10 Salmonella Typhimurium strains,2 Salmonella Pullorum/Salmonella Gallinarum strains and 36 unset salmonella strains accounted for86.01%,2.92%,0.58%and 10.50%of salmonella isolates,respectively.It was found that Salmonella Enteritidis was the main serotype of Salmonella in the production chain of laying hens in Handan area.Gre（including Gre A and Gre B）is a transcription elongation factor,which is widely found in Gram-negative bacteria.Previous studies have found that Gre factor regulates the pathogenicity of Francisella tularensis and Yersinia Pestis,and can affect the pathogenicity by regulating the expression of hil D gene in Salmonella Typhimurium.To investigate the effects of Gre factor on pathogenecity of Salmonella Enteritidis,in this study,a greAgreB double gene deletion strain and a trans-complement strain were constructed by homologous recombination of Salmonella Enteritidis ATCC13076 as a parent strain.The environmental adaptability test showed that the greAgreB double deletion had no significant effect on the temperature sensitivity and resistance to high concentrations of Na Cl of Salmonella Enteritidis.However,the greAgreB double deletion resulted in delayed logarithmic phase of Salmonella Enteritidis and significantly reduced the resistance to acid and alkali environment and SDS.At the same time,the biofilm formation ability and motility of the bacterium were significantly reduced.However,the greAgreB deletions significantly increased the resistance of Salmonella Enteritidis to H₂O₂,suggesting that Gre factor multidirectionally regulates the environmental stress of Salmonella Enteritidis.Cell infection assay showed that greAgreB double deletion reduced the ability of Salmonella Enteritidis to invade cells and multiply intracellular.Infection of chicks showed that greAgreB deletion significantly reduced the pathogenecity of Salmonella Enteritidis in subcutaneous infection,and the bacterial load of the greAgreB deletion strain in liver,spleen and lung of chickens was lower than that of the wild strain,further demonstrating that Gre factor deletion significantly affected the pathogenecity of Salmonella Enteritidis.In order to analyze the molecular mechanism of Gre factor affecting the pathogenicity of Salmonella Enteritidis,transcriptome sequencing（RNA-seq）was performed on the parent strains andΔgre AΔgre B deletion strains of Salmonella Enteritidis.The results showed that 316 genes were differentially expressed by the greAgreB deletion,accounting for 7%of the total encoding genes in S.enteritidis,of which 179 genes were up-regulated and 137 genes were down-regulated.Differentially expressed genes mainly concentrated in metabolic pathway,accounting for 71%of KEGG genes.Moreover,the differentially expressed genes were mainly distributed in40 predictive operons（more than 2 genes）,which were involved in bacterial movement,adhesion and metabolism of various substances.These results suggest that Gre factor is involved in the regulation of virulence of Salmonella Enteritidis by regulating the transcription and expression of phenotypic related genes.In conclusion,Salmonella Enteritidis is the prevalent serotype of Salmonella in Handan and its surrounding areas.Gre factor mainly regulates the environmental stress ability of Salmonella Enteritidis through the transcriptional expression of genes related to the metabolic pathway of Salmonella Enteritidis,and the loss of Gre factor reduces the pathogenecity of Salmonella Enteritidis to cell infection and chicks.This study provides a novel vaccine or drug candidate target for the preventive treatment of Salmonella Enteritidis.