Virulence Related Function Analysis Of Hfq And Small Non-coding RNA IsrE, STnc640of Salmonella Enteritidis

Small non-coding RNA(sRNA) is a new discovered factor which can regulate gene expression at post-transcriptional level in bacteria. Salmonella species are enterobacterial pathogens that have been well investigated with respect to virulence mechanisms, microbial pathogenesis and the pathways of gene expression and metabolism. Salmonella has also become a kind of model organism for RNA-mediated regulation in recent years. sRNAs play an important role in the regulation of metabolism and pathogenicity of Salmonella. However, the target genes of most sRNAs are still unknown and their functional characterization has been lagging behind. In this study, we focused on the virulence related function of sRNA chaperone Hfq, Hfq-dependent sRNA IsrE and STnc640in Salmonella Enteritidis.1. The regulatory function of sRNA chaperone Hfq in virulence of Salmonella EnteritidisSalmonella Enteritidis is an important intracellular pathogen which could infect human being and animals to cause enteritis or systemic disease.The bacterial sRNA chaperone protein Hfq can mediate the binding of small non-coding RNA to target mRNA to facilitate post-transcriptional gene regulation in bacteria. In this study, hfq deletion mutant strain50336△hfq was constructed by X-Red recombination system and the complemented strain50336△hfql/phfq was constructed by transforming fusion plasmid pBR322-hfq into50336△hfq. Then the expression of major fimbrial subunits sefA, bcfA,fimA, safA, stbA, sthA, csgA, csgD, pegA, out membrane protein ompD and flagella subunit fliC was analyzed by qRT-PCR. The results indicated that the mRNA level of sefA and ompD was increased about14folds and2.7folds respectively in50336Ahfq compared to50336. Other fimbrial subunit genes and flagella subunit fliC were all down-regulated evidently in the hfq mutant. Motility assays results showed that the motile ability of50336△hfq was decreased evidently compared to50336. We also found that the adhesion ability of50336△hfq to Caco-2cells declined significantly compared to50336. The lethal dose50%(LD50) of50336,50336△hfq and50336△hfq/phfq were tested in1-day-chickens by hypodermic injection. The result showed that the LD50of the three strains were9.472×108,2.442×109and2.974×108CFU respectively. It indicated that the virulence of50336Ahfq was attenuated in chickens. From the above results, it could be inferred that Hfq plays an important role in the virulence of Salmonella Enteritidis by regulating fimbrial genes, out membrane protein ompD and flagella subunit fliC.2. The regulatory function of sRNA IsrE in virulence of Salmonella EnteritidisA small non-coding RNA IsrE is encoded on the pathogenicity island of Salmonella, the expression level of which was increased during infection in macrophage. In this study, we constructed isrE deletion mutant50336△isrE in Salmonella Enteritidis50336by X,-Red recombination system and the complemented strain50336△isrE/pisrE by transforming the fusion plasmid pBR322-isrE into50336△isrE. Growth characteristic analysis showed that the growth rate of50336AisrE was similar to50336and the result of qRT-PCR indicated that IsrE level in50336accumulated to a peak at late stationary phase. The mRNA level of virulence related factors fimbrial subunits sefA, bcfA, fimA, safA, stbA, sthA, csgA, csgD, pegA, out membrane protein ompD and flagella subunit fliC was analyzed by qRT-PCR. The results indicated that the mRNA level of fimA、sefA、stbA、sthA、csgA、csgD、ompD and fliC was decreased in50336△isrE compared with that in wide type strain50336. The expression of ompD was decreased by95%indicated that ompD may be directly regulated by IsrE. It also showed that, compared with50336, the motile ability of50336△isrE was decreased in motility assays and the adhesion ability of50336△isrE to Caco-2cells declined in adhesion assays. The lethal dose50%(LD50) of50336,50336△isrE and50336△isrE/pisrE were tested in1-day-chicken by hypodermic injection and the result showed that the LD50of the three strains were2.901×108,4.084×108and3.101×108CFU respectively. It indicated that the virulence of50336△isrE was attenuated in chickens. As IsrE level in50336△hfq was decreased to30%compared with50336, it could be inferred that, with the help of Hfq, IsrE could regulate the virulence of Salmonella Enteritidis in some degree by regulating the expression of out membrane protein ompD, fimbrial genes and flagella subunit fliC, as well as other unknown target genes.3. The regulatory function of sRNA STnc640in virulence of Salmonella EnteritidissRNA STnc640, a kind of Hfq-dependent sRNA, was first identified in Salmonella typhimurium. However, the target mRNAs and role of STnc640were still unknown. In this study, TargetRNA software analysis showed that bcfA and fimA were probably the target genes of STnc640, which was supposed that STnc640may play a role in regulating the virulence of Salmonella Enteritidis. stnc640deletion mutant strain50336Astnc640in Salmonella Enteritidis50336was constructed by X-Red recombination system and the complemented strain50336△stnc640/pstnc640was constructed by transferring the fusion plasmid pBR322-stnc640into50336Astnc640. Growth characteristic analysis showed that the growth rate of50336△stnc640was similar to50336and the result of qRT-PCR indicated that STnc640level in 50336accumulated to a peak at late stationary phase. The mRNA level of fimbrial subunits sefA, bcfA,fimA, safA, stbA, sthA, csgA, csgD,pegA, out membrane protein ompD and flagella subunit fliC was analyzed by qRT-PCR. The results indicated that the mRNA level of fimA、sefA、csgD and ompD was decreased in50336△stno640compared with that in wide type strain50336. The adhesion ability of50336△stnc640to Caco-2cells was increased compared to50336in adhesion assays. The lethal dose50%(LD50) of50336,50336△stnc640and50336△stnc640/pstnc640were tested in1-day-chicken by hypodermic injection and the result showed that the LD50of the three strains were2.901×108,2.124×108and5.054×108CFU respectively. It indicated that the virulence of50336Astnc640was attenuated in chickens. From the above results, it could be inferred that STnc640can regulate the virulence of Salmonella Enteritidis in some degree by regulating the expression of fimbrial subunit fimA, sefA, csgD and out membrane protein ompD as well as other unknown target genes. The STnc640level in50336Ahfq was decreased to2%compared with that in50336indicated the stability of STnc640was tightly dependent on Hfq.