Screening,Identification And Functional Study Of Virulence-related Target Genes Regulated By S.Enteritidis Non-coding Small RNA RyhB

Salmonella is an important foodborne pathogen,and most serotypes are pathogenic to humans and animals,which can cause a variety of different clinical manifestations of Salmonellosis.Studying on the pathogenic mechanism of how Salmonella infected the host has important practical significance for the prevention and control of Salmonellosis.Small non-coding RNA(sRNA)is a type of RNA molecule that is transcribed but not translated into protein in the genome,which can rapidly regulate gene expression at the post-transcriptional level by sensing changes in the external environment.RyhB is a kind of sRNA existed in many bacteria such as Salmonella,E.coli and other bacteria.There are two RyhB homologs encoded by Salmonella:RyhB-1 and RyhB-2,both of which are involved in many life metabolic processes such as iron metabolism,energy metabolism,nitrate metabolism,oxidative stress,acid resistance,drug resistance and so on,but there are only few reports on the regulation of virulence-related target genes.In this study,we constructed a Simulated Intestinal Environment in vitro(SIE)model to investigate how RyhB express in S.Enteritidis,and screen for the virulence-related target genes regulated by RyhB under in SIE in vitro.Furthermore,we demonstrated regulatory mechanisms of RyhB interacted with its target genes and how RyhB affected the pathogenic mechanism of S.Enteritidis.The specific research contents are as follows:1.Low iron,hypoxia and Simulated Intestinal Environment affect the expression of RyhB-1 and RyhB-2Hypoxia and iron deficiency are two important environmental stresses which S.Enteritidis and other intestinal bacteria have to face.To explore the effects of low iron,hypoxia and imulated intestinal environment on RyhB expression and their relationship betwween these two sRNA homologs in Salmonella,we detected the growth of S.Enteritidis SE50336 wild type strain,ryhB-1 deletion mutant strain,ryhB-2 deletion mutant strain,ryhB-1/ryhB-2 deletion mutant strain,and their expression level of RyhB-1 and RyhB-2 in low iron,hypoxia,simulated intestinal environment condition.The result showed that,compared with WT cultured in LB broth aerobically,all Salmonella strains grew slowly under low iron,hypoxia,and simulated intestinal environment culture conditions;under the same culture conditions,single or double ryhB deletion affected bacterial growth.What' more,low iron,hypoxia and simulated intestinal environment all induced significantly RyhB-1 and RyhB-2 expression,especially in the simulated intestinal environment,the expression levels of the two RyhB are 70,60 times higher than that in the conventional culture conditions respectively.It indicated that RyhB-1 and RyhB-2 played an important role in respond to the above adverse environment in Salmonella.Besides,single ryhB deletion could lead to an increase in the expression of another RyhB homolog,suggesting that there is an expression complementation between the two RyhB homologs.2.Screening target genes regulated by RyhB under simulated intestinal environment in vitroIn order to study the role of regulation of RyhB when S.Enteritidis naturally infection of host,we detected the transcriptome change of wild type strain,ryhB-1 deletion mutant strain,tyhB-2 deletion mutant strain,ryhB-l/ryhB-2 deletion mutant strain under simulated intestinal environment in vitro by RNA-seq,and screened target genes regulatd by RyhB under simulated intestinal environment in vitro.RNA-seq data showed us that there were 496,250,553 Different Expression Genes(DEGs)in ryhB-1 deletion mutant strain,ryhB-2 deletion mutant strain,ryhB-l/ryhB-2 deletion mutant strain when compared with WT respectively.Among these DEGs,there were 188 genes singularly regulated by RyhB-1,110 genes singularly regulated by RyhB-2,while 363 genes together regulated by RyhB-1 and RyhB-2.14 DEGs were selected to further verified their expression under SIE in vitro by qRT-PCR.The results showed that their expression trends were consistent between both different technology,which indicated that the transcriptional analysis data are highly reliable and can continue to be analyzed further.GO analysis showed that the molecular functions of DEGs were mainly enriched in cofactor transport activity,heme transport and ATPase activity,while biological processes and cell components were mainly enriched in cofactor transport and heme transport,protein complex biosynthesis and assembly and other functions.KEGG analysis showed that the DEGs were mainly enriched in nitrate metabolism pathway,two-component system,signal transduction and bacterial invasion of epithelial cells and other virulence-related genes.3.Regulatory mechanism of RyhB-1 and RyhB-2 on virulence-related target genesRNA-seq and qRT-PCR data revealed that RyhB-1 and RyhB-2 up-regulated sipA and sopE expression,which encoded the type III secretion system effector proteins,and repressed ssal and sseA expression,which were involved in Salmonella survival and replication in macrophages.To further investigate the regulatory mechanisms of RyhB-1 and RyhB-2 on the above target genes,we predicted the interaction site between RyhB and the above target genes by bioinformatics,and revealed their regulatory mechanism(activation or inhibition)using a dual plasmid fluorescent system.The results showed that RyhB-1 and RyhB-2 directly acted with the 5'UTR of sipA mRNA,which made the closed Ribosome Bind Site(RBS)in 5'UTR of sipA mRNA exposed and promoted the translation process;RyhB-1 and RyhB-2 could also interact with ssal mRNA directly by incomplete complementary base pairing to repress ssal mRNA translation.However,the expression level of sopE and sseA genes were indirectly regulated by RyhB-1 and RyhB-2.4.RyhB-1 and RyhB-2 regulate Salmonella invasion,intracellular survival and replicationTo further demonstrate the role of RyhB on S.Enteritidis pathogenicity,a MEAT(Murine Ex vivo Anaerobic Tissue,MEAT model)model was made and proved that the ability of S.Enteritidis invasion mouse intestinal epithelial cells was attenuated after ryhB deletion.So we have a hypothesis that after naturally infection of the host,S.Enteritidis RyhB-1 and RyhB-2 are induced,and interacted with the 5'UTR of sipA mRNA to activated it translation,which affects the ability of S.Enteritidis to invade epithelial cells.Survival assays in macrophages showed that ryhB deletion resulted in a decrease in the viability of S.Enteritidis in macrophages,but increased its replication capacity,suggesting that RyhB-1 and RyhB-2 regulates the survival and replication of S.Enteritidis in macrophages mainly by direct repressed the expression level of a effector protein Ssal,which are involved in the survival and replication in macrophages.