Screening Of Transcribed Sequences From Salmonella Enterica Serovar Enteritidis In Different Sources Of Macrophages By Selective Captured Of Transcribed Sequnecs (SCOTS)Technique

Salmonella enterica serovar Enteritidis (SE) is an important foodborne and zoonotic pathogen which is linked to human infection. Consumption of SE-contaminated poultry products is one of the major risk factors for outbreaks of human SE infections. Infection of SE can cause gastrointestinal diseases represented by enterocolitis in humans. Generally, this type of infection is restrictive and cleared within3-5days. However, chickens less than2week old showed obvious clinical symptoms and a high level of mortality when infected with SE. And invasion of laying hens by SE caused systemic infection and contamination of eggs. Currently, the study of pathogenesis of SE is still in primary stage. Therefore, searching for potentially virulent genes is very important to reveal the pathogenesis of SE and develop therapeutic measures to the disease. In this study, the selective captured of transcribed sequences (SCOTS) technology was used to screen and identify genes expressed by SE-infected chicken and murine macrophages. This study provided the information for us to understand the pathogenesis of SE infection of different hosts.1. Identification of SE genes expressed during the infection of macrophages from different sources by selective capture of transcribed sequences techniqueIn order to compare and analyze the invasion ability and proliferation trend of SE strain C50041in two different types of macrophages, we chose murine macrophages RAW264.7and chicken macrophages HD-11as cell models. The result showed that the invasion ability of SE in HD-11was higher than RAW264.7. And there was significant difference in the proliferation ability at different times (eg:5hr post phagocytosis) between RAW264.7and HD-11. Then the selective captured of transcribed sequences (SCOTS) technology was used for screening the expressed sequences at2hr after infection with SE. The functions of obtained sequences were analyzed by sequencing and Blast analysis. We obtained58and54transcribed sequences from SE-infeced chicken macrophages and murine macrophages respectively. These expressed genes were involved in various aspects including virulence, metabolism, environmental adaptation, molecular synthesis/degradation, regulator system, transport system and unknown functions. The study showed that most of the captured sequences were obvious different from SE-infected RAW264.7cells to HD-11macrophages except only two sequences. The results provided information for us to understand the pathogenesis of SE infection to different hosts.2. Differential expression of infection-related factors from SE strain C50041detected by real-time fluorescent quantitative PCR in in vivo and in vitro condition.According to the sequences screened out by SCOTS technology,12genes were selected randomly from58sequences identified during SE infected chicken macrophags HD-11(invJ、 ycaM、bigA、SEN4299、SEN3610、SEN0988、nlpB、SEN2555、SEN0815、SEN2967、stdB and yiaG), and12genes from54sequences identified during SE infected murine macrophages RAW264.7(dnaG、cbiK、rbfA、copS、rtcR、rfaQ、rseB、secG、spvB、flgK、lpfC and phoB) were selected to analyze their differential expression in different conditions by quantitative real-time PCR(qRT-PCR) analysis. Then qRT-PCR analysis was performed with SYBR(?) green and analyzed with2-ΔΔCT for gene expression comparison between2hr post infection and5hr post phagocytosis, and the difference of transcription level of mRNA was detected in in vivo and in vitro conditions. The result showed that SCOTS technology was reliable because all the genes were expressed in both macrophages and most of these genes were up-regulated apparently during the infection compared to cultivation in vitro. The genes screened from SE-infected HD-11showed higher expression level at5hr post phagocytosis compared with2hr post infection of HD-11cells, while the expression level showed no significant difference in RAW264.7at both time points. However, the genes identified from SE-infected RAW264.7showed tiny difference at the two time points following infection to RAW264.7, and the expression level was significantly reduced in HD-11compared with RAW264.7, particularly at5hr post phagocytosis. Moreover, some genes were up-regulated to20-fold during the infection process, and some even up to100-folds. The genes screened out which have significant difference in different sources macrophages help us to reveal pathogenesis of SE in different host.