Transcriptome Analysis Of Salmonella Typhimurium And Functional Study Of Polym Yxin B Tolerance-Related Genes

Salmonellosis is an infectious disease caused by Salmonella infection,which is common to humans and animals and causes serious harm to the aquaculture industry.Salmonellosis has also become a global public health problem due to the rising infection rate in the population.Antimicrobial peptides are a class of polypeptides or proteins produced by animals and plants with broad-spectrum antimicrobial activity.In this experiment,the gene expression differences of Salmonella typhimurium after polymyxin B stimulation were analyzed by transcriptome sequencing technology,and the differential genes were annotated and clustered.According to the gene function,the target genes were selected.Using Red recombination technology,Salmonella typhimurium was used as the parental strain to construct the relevant gene deletion strains and their complementary strains,and their growth characteristics,polymyxin B resistance were studied.Biological characteristics such as receptivity,antioxidant capacity,acid resistance,cell infection and virulence in vivo were determined.The results of RNA-seq analysis showed that there were 525 significantly different genes in Salmonella typhimurium stimulated by polymyxin B.including 297 up-regulated genes and 228 down-regulated genes.GO functional clustering showed that the Salmonella differential genes stimulated by polymyxin B mainly focused on non-membrane-bounded organelle,ribonucleoprotein complex,macromolecule localization,protein transport,structural molecule activity and other functions,suggesting that the main action sites of polymyxin B and Salmonella are in the cell membrane,and the related genes are involved in the synthesis and transport of intracellular proteins in the KEGG pathway;The analysis results showed that flagellar assembly,cationic antimicrobial peptide resistance,glyoxylate and dicarboxylate metabolism,and biosynthesis of siderophore group non-ribosomal peptides were significantly enriched.It can be seen that the main action pathways of polymyxin B are cell movement,immune regulation and small molecule synthesis.According to the sequencing results and literature reports,19.target genes were selected for subsequent studies.Red recombination technology is widely used in the construction of Salmonella gene deletion strains.The target gene sequence was determined according to the whole genome sequence of S.Typhimurium ATCC 14028s published by NCB1.The conditions for gene deletion were explored as follows:(1)the concentration of targeting fragments was 500-800 ng;(2)the concentration of L-arabinose was 30 mmol/mL and the OD600 value was about 0.6-0.8 when electroporating the linear targeting fragments;(3)the voltage was 2 kV when electroporating the linear targeting fragments;(4)the strain was resuscitated in a shaker at 42℃ for 12 h when the chloramphenicol gene was eliminated.Finally,the following gene deletion strains were successfully constructed:SMΔsapB,SMΔsapC,SMΔyejE,SMΔyejF,SMΔompC,SMΔompW,SMΔSTM1485,SMΔSTM2759,SMΔyjeA,SMΔsufC,SMΔsufD,SMΔmarA,SMΔmgtC,SMΔpmrD,SMΔydhC,SMΔmgtC,SMΔscsA,SMΔtetR,SMΔexbB.And successfully constructed their complementary strains.The results of biological function study on 19 gene deletion strains constructed above showed that compared with the parental strains,the deletion of all genes did not affect the growth of Salmonella;the resistance of SMΔyejE,SMΔompW,SMΔmarA,SMΔpmrD to polymyxin B was significantly weakened;the resistance of SMΔsapC and SMΔmarA to acidic environment was reduced.It was proved that yejE,ompW,marA and pmrD genes were related to polymyxin B resistance,sapC and marA genes were related to acid tolerance of Salmonella,and marA genes were also related to acid tolerance and antimicrobial peptide resistance.The intracellular viability of SMΔyjeA,SMΔsapB,SMΔsapC and SMΔmgtC was decreased,the hepatic and splenic bacterial loads of mice injected with SMΔyjeA and SMΔsapB were decreased,and the in vivo survival time was shortened,indicating that the deletion of yjeA and sapB genes affected the virulence of Salmonella both in vivo and in vitro.In summary,this study conducted a transcriptomic analysis of Salmonella stimulated by polymyxin B and selected target genes,constructed gene-deleted strains using Red recombination system,and determined the biological characteristics of gene-deleted strains in vitro and in vivo.Genes related to antimicrobial peptides and acid tolerance of Salmonella and virulence-related genes were preliminarily screened to further elaborate the pathogenesis of Salmonella.The development of mechanism and genetically engineered vaccines provides data support.