高盐环境下鼠伤寒沙门氏菌差异糖蛋白富集及组学分析

Enrichment and omics analysis of differential glycoproteins in Salmonella enterica serovar Typhimurium under high salinity environment

【背景】沙门氏菌是一种革兰阴性肠道病原菌,主要依靠Ⅲ型分泌系统(type Ⅲ secretion systems,T3SSs)来产生与致病性相关的效应蛋白。其中沙门氏菌致病岛(Salmonella pathogenicity island,SPI)区域是关键的基因区域。高盐浓度条件可以诱导SPI-1上效应蛋白的表达。【目的】探究在高盐浓度条件下鼠伤寒沙门氏菌(Salmonella enterica serovar Typhimurium)糖蛋白的差异表达情况,寻找有意义的效应糖蛋白。【方法】将鼠伤寒沙门氏菌在普通培养基和高盐培养基中培养,收集菌体并超声裂解,提取蛋白后,用肼偶联法富集糖蛋白并用胰酶酶解,通过二甲基标记定量及LC/MS定量蛋白质组学方法进行糖蛋白的定量,用Thermo Proteome Discoverer 2.2软件对标记蛋白进行定性及定量分析。【结果】质谱结果显示,高盐环境中,沙门氏菌有19个糖蛋白的表达发生显著改变,其中,上调蛋白10个,最为显著的是ompC基因编码的外膜孔蛋白;下调表达糖蛋白9个,最为显著的是yjgF基因编码的翻译起始抑制因子。【结论】根据定量蛋白质组学分析,沙门氏菌在高盐环境下有多种重要糖蛋白的表达水平发生显著改变,这一结果对研究沙门氏菌SPI-1产生的效应糖蛋白表达情况以及寻找沙门氏菌致病机制有重要意义。

[Background]Salmonella is a kind of Gram-negative intestinal pathogen that relies primarily on type Ⅲ secretion systems(T3SSs)to produce pathogenicity-associated effector proteins.The Salmonella pathogenicity island(SPI)is a key genetic region.The conditions of high-salt can induce the expression of effector proteins on SPI-1.[Objective]In order to explore the differential expression of glycoprotein of Salmonella enterica serovar Typhimurium under high-salt concentration,and to find meaningful effector glycoprotein.[Methods]Salmonella enterica serovar Typhimurium was cultured in common medium and high-salt medium,and then the cells were collected.The glycoproteins were enriched by hydrazine coupling method after ultrasonication and protein extraction.After the enzymatic hydrolysis,the glycoproteins were quantified by dimethyl-labeled quantitative method,and the labeled proteins were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry.[Results]Mass spectrometry results showed that the expression of 19 glycoproteins in Salmonella significantly changed in the high salt environment.Among these,10 glycoproteins were up-regulated,the most notably one was the outer membrane porin encoded by ompC.Nine glycoproteins were down-regulated,the most notably one was translation initiation inhibitor encoded by yjgF.[Conclusion]The expression levels of many important glycoproteins in Salmonella have significant changes at high-salt environments.This study has important significance in investigating the expression of effector glycoprotein produced by Salmonella SPI-1 and searching the pathogenic mechanism of Salmonella.