Regulatory Pathways Of SRNA SaaS On The Adhesion Behavior Of Salmonella

Salmonella is a food-borne pathogen that endangers public health worldwide.One-third of salmonellosis is caused by Salmonella Enteritidis.The contamination of Salmonella in food is mainly due to the cross-contamination caused by the biofilm formed on the environment and equipment during food processing.The bacterial cells are embedded in the biofilm to make them more viable and adaptable to the environment.The presence of biofilm poses a great risk of contamination in food processing.Biofilm formation is divided into 6 stages.Flagellar and fimbriae/pili,which play a major role in the initial adhesion phase.During the maturity of biofilms,extracellular polymeric substances(EPS)play a major role.The synthesis of these structures and substances can reflect the adhesion potential and adhesion state of the biofilm.At the molecular level,biofilm formation is regulated by a variety of regulatory pathways.There are core mRNAs in the pathways that encode core regulatory factors,which affect biofilm formation by controlling the inhibition or activation of downstream gene expression.In addition to regulation at the transcription,regulation at the post-transcription also has an important effect on the biofilm formation.Non-coding small RNA(sRNA)is a kind of important post-transcriptional regulator.This paper takes a newly excavated sRNA SaaS as the research object.Studies have shown that this sRNA can increase the adhesion potential and biofilm formation of S.Enteritidis.In order to further confirm the molecular regulation mechanism of SaaS to regulate biofilm formation,the laboratory constructed a mutant strain with specific deletion of sRNA SaaS(?SaaS)based on the S.Enteritidis wild type strain(WT).At 37?,trypticace soy broth(TSB)was used as the medium,and the 96-well cell plate made of PVC was used as the adhesion surface.The biofilm was cultivated to determine its formation rule,and the mature biofilm cells were used as samples to determine the transcriptiome and proteome.The effect of SaaS on biofilm formation-related regulatory pathways and the core target mRNAs in the pathways that SaaS may act on were determined by transcriptomics and proteomics correlation analysis.The molecular regulation mechanism of SaaS on biofilm formation is further revealed.The specific research contents are as follows:1.Analysis of sRNA SaaS and biofilm formation regularity of the strains.The genomic of the WT was determined.Locate the position of SaaS in this genome,and this genome was used as the reference genome for the subsequent transcriptome and proteome.The chromosome genome was 4,679,739 bp,and the GC content was 52.17%.4,660 coding sequences,106 structural RNAs(22 rRNAs and 84 tRNAs)and 14 gene islands(GI)were annotated.The SaaS and 4 other novel sRNAs were aligned into 18 food-related bacterial genomes to determine their conservation.The TSB was used as the culture medium at 37?,and the PVC 96-well cell plate was used as the adhesion surface to cultivate the WT and ?SaaS biofilm.During the cultivation,part of the cultured cell plates was taken out every 12 hours,and the crystal violet staining test and the live cell count test in the biofilm were carried out to determine the biofilm formation rule of the two strains.By comparison,the effect of SaaS on the biofilm formation ability was shown.The test results showed that:SaaS was highly conserved in Salmonella,indicating that it may have important biological functions.SaaS is located on the positive strand complementary to the 3'-UTR of the hilD encoding gene in the WT genome,and the complementary region of SaaS sequence overlaps with 5 nucleotides at the 3' end of hilD mRNA.SaaS sequence in Rfam database search showed that it overlapped with partially CsrA protein binding site.SaaS may be related to hilD expression and CsrA protein regulation.When the two strains were cultured for 48 hours under the set conditions,the biofilm was mature and the total amount of biofilm formation was the largest.The results of crystal violet staining showed that the total amount of biofilm formation of ?SaaS was significantly increased relative to WT.During the culturing of the biofilm,the number of viable cells in the biofilm formed by ?SaaS was always about 1 Log CFU/ml more than that in the biofilm formed by WT.The absence of SaaS significantly increased the amount of biofilm formation,and the number of living cells in the biofilm increased,which indicates that SaaS has an inhibitory effect on biofilm formation.The biofilm cells in the mature period were collected for subsequent transcriptome and proteome determination.2.The effect of SaaS on the transcriptome of adherent cells of S.Enteritidis.Taking mature biofilm cells cultured in Chapter 1 as samples.The total RNA from them were extracted for quality inspection and performing on the machine.The expression levels of the measured mRNAs in the two strains and the relative expression multiple of?SaaS relative to WT were calculated to determine the differentially expressed genes(DEGs)in the two strains.GO function classification and KEGG pathway enrichment analysis were performed on the DEGs,and important regulatory pathways related to biofilm formation were selected.The results showed that the coverage of the detected genes in the RNA samples of the two strains accounted for more than 97%of the reference genome.There were 725 DEGs in the two groups of samples,including 253 up-regulated genes and 472 down-regulated genes.The DEGs were enriched in a total of 110 regulatory pathways,including flagellar assembly,biofilm formation,bacterial chemotaxis,two-component system,bacterial secretion system and quorum sensing.The above 6 pathways will also be the focus of follow-up analysis.The DEGs involved in bacterial chemotaxis and flagellar assembly system were generally down-regulated.The core regulatory gene csgD of EPS synthesis in the biofilm formation pathway was significantly down-regulated,well the genes csgAB encoding subunit protein of curli were significantly up-regulated.This indicates that the amount of curli synthesis may increase.This may be the direct cause of the increase in biofilm.SaaS may inhibit flagella expression while promoting biofilm formation.3.The effect of SaaS on the proteome of S.Enteritidis adherent cells and its target mRNA predictionTaking mature biofilm cells cultured in Chapter 1 as samples.The total proteins from them were extracted for quality inspection and performing on the machine.The expression levels of the measured proteins in the two strains and the relative expression multiple of?SaaS relative to WT were calculated to determine the differentially expressed proteins(DEPs)in the two strains.GO function classification and KEGG pathway enrichment analysis were performed on the DEPs.Correlate the DEGs and DEPs involved in the six important regulatory pathways selected in Chapter 2.All genes in the six pathways,hilD and csrA mRNA were predicted for SaaS target genes,and the core mRNA most likely to be directly regulated by SaaS was selected among all predicted target genes.The results showed that there were 490 DEPs in the two groups of samples,including 97 up-regulated proteins and 393 down-regulated proteins.The DEPs were enriched in a total of 107 regulatory pathways.Combining transcriptome and proteome data,flagellin,type ? fimbriae(T1F)proteins and these coding genes that played a major role in the initial stage of adhesion were significantly down-regulated.The expression of type ? pili(T4P)protein was significantly up-regulated.EPS synthesis regulation gene csgD mRNA,part of cellulose and colain acid synthesis-related mRNAs were significantly down-regulated.The expression levels of the curlin subunit encoding genes csg/AB mRNA and small subunit protein CsgA were significantly up-regulated.Using IntaRNA software,294 possible target genes of SaaS were predicted.Five genes including cheA,csrA,csgA,hilD,and adrA,are likely to be target mRNAs directly affected by SaaS.