Salmonella Produce MicroRNA-like RNA Fragment Sal-1 In The Infected Cells To Facilitate Intracellular Survival Via Attenuating INOS Induction

Salmonella is the main pathogen which causes food-borne illness in the world.As a zoonotic intestinal pathogen,Salmonella has developed a sophisticated machinery to evade host innate immune defense and promote survival in the infected cells.Previous studies on the interaction of host-pathogens during Salmonella infection mainly focused on the various virulence factors which encoded and secreted by their pathogenicity islands and type III secretion systems(T3SSs),especially on the regulation of virulence factors expression.In recent years,the regulation role of non-coding RNAs(ncRNAs)in the immune response has been widespread concern.In the process of pathogens infction,the bacterial source ncRNA can regulate the expression of the virulence factors in the course of the bacterial response to the environmental stress of the host,and then help the bacteria adapt to the environment and complete its survival and reproduction.At present,the exploration of ncRNA during bacterial-host interaction is still in the initial stage.In this paper,we find a new mechanism for the regulation of host immunization by non-coding RNAs from Salmonella in the process of infection.In the model of Salmonella infection,the small RNA fragments from Salmonella were found in the infected cells by Solexa.The size of them was 19-24nt,which was similar to the eukaryotic microRNAs(miRNAs),and the abundance of them was close to the cell endogenous miR-107.We speculated that the infection process produced miRNA-like small RNA fragments,which were from Salmonella and distinguished from the longer ncRNA encoded by the bacteria itself.According to the Solexa results,we chose five RNA fragments with the highest abundance and detected their expression using qRT-PCR.The results showed that these small RNA fragments could only be produced after the bacteria infected cells,indicating that the production of these small RNA fragments depended on host cells.Then we focused on Sal-1,one such fragment with the highest copy number in the infected cells,and analyzed its biogenesis using bioinformatics.Sequence analysis showed that Sal-1 was derived from Salmonella 5'-leader of the ribosomal RNA transcript and had a 'stem-loop'structure-containing precursor which similar to eukaryotic pre-miRNAs.We synthesized the sequence of this precursor and inserted it into the eukaryotic expression vector,then transfected the plasmid into host cells.After transfection,the Sal-1 fragment was detected in the cells,suggesting that the specific 'stem-loop'structure could be cut into miRNA-like fragments in cells.Based on canonical and non-canonical pathways of miRNA biogenesis,we then identified the proteins which involved in the maturation process of Sal-1 using RNAi.Unlike the eukaryotic classic miRNA maturation process,processing of Sal-1 precursors to mature Sal-1 was dependent on host cell Argonaute 2(Ago2)but not Dicer.Further research on plasmid-transfected and Salmonella infected cells showed that Ago2 participated in the whole process of Sal-1 maturation.We were also interested in the transport mechanism of this RNA fragments,and the results showed that Salmonella can package their non-coding RNAs in outer-membrane vesicles(OMVs)and deliver them into host cells.Functionally,modeled on miRNA function studies,we looked for potential target genes of Sal-1 in host cells using several experiments and finally found that Sal-1 targeted cellular inducible nitric oxide synthase(iNOS)and regulated it in a non-classical miRNA manner,leading to attenuation of host cell iNOS-mediated anti-microbial capacity and enhancement of Salmonella intracellular survival.Depleting cellular Sal-1 or expressing a functionally active iNOS mutant not targeted by Sal-1 renders the Salmonella bacteria less resistant to the host defenses in vitro and in vivo.In summary,Salmonella-infected host cells contain Salmonella-encoded functional small non-coding RNA fragment Sal-1,which involved in the regulation of the host immune system and thus creating a microenviroment conducive to the survival of bacteria.This RNA fragment may constitute a novel virulence factor or regulatory factor which will reveal the pathogenic mechanism of pathogens infection,and will help us control various diseases caused by pathogenic microorganisms.