| Salmonella species are widely-existing food-borne pathogen that infect humans and animals,causing typhoid,gastro-intestinal inflammation,sepsis and other diseases.Due to the abuse of antibiotics,its increasingly serious resistance to antibiotics,and the study of its drug resistance mechanism is of great significance to food safety and livestock industries.Salmonella strains have different mechanisms of resistance to different types of drugs,and clinically isolated strains are mostly multidrug-resistant(MDR)strains,so individual drug resistance mechanisms cannot be specifically studied.Therefore,in this study,a single drug resistance strains were selected to study the relationship between CRISPR/Cas system and drug resistance in Salmonella,and to explore the resistance mechanism of Salmonella.Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is present in most bacterial genomes,CRISPR and CRISPR-associated protein genes downstream of its sites constitute the CRISPR/Cas system.This system is the main immune system of bacterias and has the functions of recognizing and processing exogenous plasmids and bacteriophages.Currently,studies have shown that the CRISPR/Cas system is related to the resistance of bacterias.In this study,ciprofloxacin-resistant strains were selected in vitro,The CRISPR/Cas system sequence alignment,the differential expression analysis of cas mRNA expression,and the analysis of Cas protein expression difference were performed between the resistant and standard strains.Real-time quantitative PCR(RT-qPCR),tandem mass tag(TMT)and parallel reaction monitoring(PRM)target protein verification were used to study the ralationship between CRISPR/Cas system and FQs resistance in Salmonella.The main research contents and results are as follows:1.By matching the CRISPR/Cas system of S.typhimurium ATCC 13311,a CRISPR sequence was detected at the 3013863 bp-3014930 bp position of the genome.Eight cas sequences were found downstream of the sequence,followed by cas2,cas1,cas6,cas5,cse4,cse2,casA and cas3.The CRISPR/Cas system was identified as Type I-E.2.The CRISPR/Cas system sequences of FQs resistant strains were 273 single-base mutations and 20-base fragment insertions or loss were found on the system compared with those of the standard strain.3.The mRNA expression of the 8 cas genes were found different between resistant and standard strains using RT-qPCR.Among them,cas3,cas5 and cas6 expression were downregulated,cse2 and cse4 expression were upregulated,cas1 and cas2 expression were relatively stable.4.The expression of Cse4 protein was found significantly upregulated by using TMT proteomics quantitatively combined with PRM targeting verification technology.5.The proteomics data were analyzed and processed,and the differential proteins were screened at a 2-fold change threshold(Fold change ?2 or ?0.5),p<0.05,the bioinformatics analysis was performed.The results showed that the expression of a total of 318 proteins were screened out differential,of which 159 were upregulated and 159 were downregulated,involving efflux pump-associated proteins,outer membrane proteins,two-component related proteins and pathways,bacterial chemotaxis-related proteins and pathways,etc.The selected 15 differential proteins were verified by the PRM targeting protein technology and successfully quantified 13 proteins and matched with the TMT results.In this study,FQs-resistant strains were selected in vitro.And the differences between the standard and the resistant strains CRISPR/Cas system were analyzed from the gene sequence,transcription level and protein level.At the same time,several proteins and pathways related to drug resistance were screened by using TMT combined with PRM technology.To provide a theoretical basis for studying the relationship between Salmonella CRISPR/Cas system and drug resistance,and laid the foundation for further study on the resistance mechanism of Salmonella. |
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