Regulation Of Non-coding Small RNAs RyhB On Virulence-related Genes InvH And StdA In Salmonella Enteritidis

Salmonella enterica serovar Enteritidis is one of the most important pathogens causing non-typhoid Salmonella infection.Salmonella colonizes the intestinal and reproductive tracts of adult poultry for long periods,causing a recessive infection.And it is continuously discharged by infected animals for a long time,which brings huge economic losses to the aquaculture industry.After infection,Humans suffer from gastroenteritis and even death in severe cases.As a zoonotic pathogen,the research of pathogenic mechanism of Salmonella Enteritidis has gradually attracted people's attention.Non-coding small RNAs are a kind of short-length RNA molecules that located in the intergenic region without encoding proteins,having various regulatory functions.As the global regulatory RNAs,RyhB-1 and RyhB-2 regulate iron metabolism,nitrate metabolism,acid resistance,adhesion and invasion ability,anti-oxidative stress and so on,which are significant to virulence and pathogenicity of Salmonella.Salmonella Enteritidis encodes two RyhB paralogs,RyhB-1 and RyhB-2,which share a 33 bp conserved sequence.They regulate Salmonella virulence by regulating the same or different virulence factors.stdA is the main subunit of Salmonella std fimbriae gene cluster.It encodes StdA fimbriae protein,which is closely related to the adhesion function of Salmonella.invH is not only the outer ring structure of the three-type secretion system(TTSS),but also the effector secreted by TTSS-1,which contributs to Salmonella invasion function.Our previous data of transcriptome sequencing showed that the deletion of RyhB led to a significant down-regulation of mRNA levels of invH and stdA under simulated intestinal environment.We speculate that RyhB promote the expression of invH and stdA.To explore whether RyhB regulate Salmonella pathogenicity by promoting the expressions of invH and stdA,we verified the mRNA and protein levels of invH and stdA by fluorescence quantitative PCR and western blot,respectively.And we tested whether RyhBs modulated Salmonella pathogenicity by regulating the virulence genes invH and stdA in vitro and in vivo.The results showed that RyhB deletion strains resulted in a significant decrease in the mRNA and protein levels of invH and stdA under simulated intestinal conditions.It indicates that RyhBs significantly up-regulate the expression of invH and stdA,which is consistent with the previous transcriptome sequencing results previously.Caco-2 cell adhesion and invasion experiment results showed that single deletion of RyhB or invH led to a significant decline of Salmonella invasion.And the invasion ability of RyhB single deletion strain was higher than that of invH single deletion strain.But the number of invasive Salmonella was decreased significantly in RyhB and invH double deletion strain compared with the invH single deletion strain,indicating that RyhB promote the invasion function of Salmonella by regulating the expression of invH.Also,the The results of adhesion experiment showed that the number of adhesive Salmonella of RyhB and stdA double deletion strain was significantly reduced compared with the stdA single deletion strain.These results demonstrated that both RyhB and stdA significantly promote the adhesion ability of Salmonella,but whether RyhB directly regulate stdA needs further research to study.The results of animal infection experiments showed that ryhB-2 and invH double deletion strain resulted in a significant decrease in the number of Salmonella colonization in the spleen,liver and small intestine on the 5th and 7th days of infection compared with the invH single deletion strain.And the ryhB-1/invH double deletion strain resulted in a significant decrease in the number of Salmonella colonization in liver and small intestine on the 7th day compared with the invH single deletion strain,indicating that RyhB may promote Salmonella colonization in chick by regulating invH at the early stage of infection.On the other hand,the stdA single deletion strain showed the lowest number of Salmonella colonization.The RyhB and stdA double deletion strain resulted in a significantly increased at the number of Salmonella colonization in spleen,liver and small intestine compared with stdA single deletion strain.The results indicate that as a virulence gene,stdA plays an important role in promoting Salmonella colonization in vivo,but whether RyhB can promote bacteria colonization in vivo by regulating the expression of stdA needs to be further studied.As a global regulatory protein modulating iron metabolism in bacteria,Fur deregulates RyhB in low iron environment and promotes RyhB to inhibit the expression of iron storage-related proteins to maintain the balance of cellular iron balance.Whether Fur affects the regulation of RyhB on target genes,invH and stdA,and the regulation of Salmonella pathogenicity has not been reported.In this paper,firstly,the fur,fur/ryhB-1,fur/ryhB-2 deletion strains and corresponding complemented strains were constructed by ?-Red homologous recombination technology.Secondly,the mRNA levels of virulence genes invH and stdA of strains were detected under simulated intestinal conditions by using qRT-PCR.Thirdly,the Caco-2 cell adhesion and invasion assay were used to explore the regulatory of Fur to RyhB on cell adhesion and invasion.The results showed that fur significantly reduced the expression of invH under simulated intestinal conditions,and promoted the expression of stdA by regulating RyhB.On the other hand,the results of cell adhesion and invasion showes that fur promotes the invasion ability of Salmonella by regulating RyhB.