Study On The Resistance Mechanism Of Salmonella Based On Proteomics And Protein Acetylation Modification

Salmonella is an important food-borne zoonotic pathogen,which can cause diseases such as gastroenteritis,typhoid fever,and sepsis,it is of greater harm to human health and animal husbandry development.Fluoroquinolones(FQs)are common drugs used treating Salmonellosis,due to the widespread and unreasonable use or even abuse of FQs in clinical treatment,which cause the resistance rate of Salmonella is constantly increasing.The resistance of Salmonella has become a critical clinical problem.Therefore,it is of great significance to study and conquer the drug resistance of Salmonella to FQs and explore its resistance mechanism.At present,the commonly used methods for studying the mechanism of bacterial resistance is to artificially induce the resistance of sensitive strains to a single drug,and obtain stable drug-resistant strains for further research.In vitro induction of ciprofloxacin-resistant strains against Salmonella typhimurium had been completed in the laboratory.In this study,two-dimensional electrophoresis and mass spectrometry techniques were used to screen the differentially expressed proteins of the resistant strains of Salmonella typhimurium to ciprofloxacin in vitro and standard strain,in order to identify the drug resistance-related proteins of Salmonella typhimurium resistant strains.The TMT marker and acetylation enrichment techniques were used for quantifying the acetylation modification of Salmonella ciprofloxacin-induced drug-resistant strains and standard strains in vitro,screened proteins with differential expression of acetylation sites,and conducted quantitative studies and bioinformatics analysis,these methods provided an important basis for further study of the relationship between protein acetylation and Salmonella resistance.The specific research contents and results are as follows:First,differential analysis of protein expression between Salmonella resistant strains and standard strains.Salmonella-resistant and standard strains were cultured to logarithmic phase in LB medium,and then prepared protein samples by sonication of bacteria,and performed two-dimensional electrophoresis.After electrophoresis,the protein spots were selected for mass spectrometry identification.25 differential expression proteins were screened between resistant and standard strains,22 proteins were successfully identified,the proteins were classified into 4 categories according to its function:5 resistance-related proteins and 2 virulence-related proteins,3 proteins related to protein synthesis and 12 proteins related to metabolism.The results indicated that the occurrence of resistance caused multiple proteins changes,suggested that these proteins were related to the development of Salmonella resistance.Second,the acetylation modification of Salmonella resistant strains and standard strains were analyzed.The in vitro induced ciprofloxacin-resistant and standard strains were used for research object.The TMT and acetylation enrichment techniques were used screening the proteins with different expression of acetylation sites after drug resistance and conducted quantitative and bio informatics analysis.The results that a total of 631 acetylated proteins were identified and involved 1259 acetylation sites.Among the quantitative acetylation sites,the modification level of 112 sites was up-regulated and the modification level of 149 sites was down-regulated compared with the standard strains.The main enrichment pathways were metabolism process of microorganism,antibiotic biosynthesis,and bacterial chemotaxis.Among the proteins with significantly different levels of acetylation,13 proteins related to bacterial resistance were screened.The results indicated that the acetylation of proteins modification involved in the emergence of resistance to Salmonella in vitro.In this study,in vitro induction of ciprofloxacin-resistant strains against Salmonella typhimurium and standard strains were used for conducting the proteomics and protein acetylation modification analyses,and screened the differential proteins and metabolic pathways related to drug resistance,the thesis laid the foundation for in-depth study of the mechanism of Salmonella.