Interaction Of Small Non-Coding RNA RyhB-1 And IsrE In The Pa Thogenicity Of Salmonella Enteritidis

RyhB is a kind of about 90 bp bacterial non-coding small RNA which could regulate gene expression in post-transcriptional level. It was reported that two RyhB homolog RyhB-1 and IsrE, which existed in the Salmonella Typhimurium and Vibrio cholerae could regulate biofilm formation, chemotaxis and acid resistance. In this study,we focused on the pathogenicity and cooperative function of RyhB-1 and IsrE in Salmonella Enteritidis.1 The genes expression of RyhB-1 and IsrE under the different environment conditions in Salmonella EnteritidisWe designed the primers of RyhB-1 and IsrE gene based on sequences in Salmonella Enteritidis, then tested genes expression when the growth of bacteria was cultured to different period at iron starvation, Simulated intestinal environment,infect with macrophage by qRT-PCR. The results showed that, at exponential phase, the expression of RyhB-1 and IsrE in iron starvation condition enhanced 2-fold compared with that in LB medium culture, while at stationary phase, the gene expression of RyhB-1 and IsrE enhanced 2.5-fold and 3-fold respectively, which indicated that the strain need efficient absorption of iron under iron starvation invironment and two sRNA could regulate iron metabolism. In Simulated intestinal environment, the expression of RyhB-1 and IsrE enhanced 3.5-fold and 3-fold respectively at exponential phase, while 1.3-fold and 1.5-fold at stationary phase. When the bacteria infected macrophage, the expression of RyhB-1 increased 2-fold and 5.5-fold at lh and 4h, while expression of IsrE increased 4.5-fold and 8.5-fold. It indicated that the gene expression of both genes must be enhanced by induced in the environment pressure. The change fold of IsrE expression is higher than RyhB-1. It was inferred that RyhB-1 and IsrE are related to virulence with different regulated mode.2 The construction of RyhB-1 and IsrE double deletion mutant strain and the complement strain in Salmonella EnteritidisThe ryhB-1 and isrE single deletion mutant strain were constructed by X-Red recombination system in previous study. The ryhB-1 and isrE double deletion mutant strain was constructed by the same system in this study. We designed the primers of ryhB-1 and isrE sequences contained with upstream and downstream non-coding sequence and restriction enzyme cutting site to construct complemented strains by transforming fusion plasmid pACYC184-ryhB-1 or pBR322-isrE into single deletion mutant 50336AryhB-1 or 50336ΔisrE. The double complemented strain of ryhB-1 and isrE was constructed by transforming fusion plasmid pACYC184-ryhB-1 and pBR322-isrE into 50336ΔryhB-1/ΔisrE. the construction of these strains that would play an important role in that we are exploring for pathogenic function of two homolog ryhB-1 and isrE in Samonella Enteritidis.3 The pathogenicity analysis of RyhB-1 and IsrE in vivo or in vitro in Salmonella EnteritidisWe tested the virulence regulation and the pathogenic differences of wild type, RyhB-1 and IsrE single or double deletion mutant strains and the complement strains by the athletic ability of bacteria, the ability of biofilm formation, the gene expression of activity-based, the invasiveness and adherence for the host body, the anti-phagocytosis assays and the survival rate in macrophages,inoculating above strains to one-day-old chickens infection and LD50 detection, mortality rate and the strains distribution in chickens.The results showed that the virulence and these function of Salmonella Enteritidis decreased in RyhB-1 or IsrE deletion mutant strain, especially in RyhB-1 and IsrE double deletion mutant strain. It indicated that two sRNAs would enhanced pathogenic of Salmonella Enteritidis.