应用iTRAQ定量蛋白质组学技术筛选无乳链球菌鱼源株与人源株差异表达蛋白

Quantitative proteomic analysis of differential proteins in Streptococcus agalactiae piscine strain and human strain using iTRAQ

以斑马鱼和巨噬细胞作为体内、外感染模型,比较无乳链球菌鱼源株GD201008-001与人源株A909的致病性差异,利用iTRAQ技术和质谱分析技术进行差异表达蛋白鉴定,以期为揭示无乳链球菌不同宿主来源株致病机制提供新思路。实验通过测定菌株GD201008-001、A909的斑马鱼半数致死量和巨噬细胞吞噬率,比较二者致病性差异;提取全菌蛋白,经iTRAQ试剂标记后进行质谱鉴定,质谱数据用软件Mascot 2.2和Proteome Discoverer 1.4进行查库(Uni Prot数据库)鉴定及定量分析,并对差异蛋白进行GO功能注释和KEGG通路分析。结果显示,GD201008-001毒力显著高于A909;通过iTRAQ分析两株菌差异表达蛋白,发现差异蛋白涉及的生物学功能较为广泛,在鉴定出的368个差异表达蛋白中,GD201008-001中上调表达蛋白193个(比值>1.5),下调表达蛋白175个(比值<0.667)。生物信息学分析预测这些蛋白主要涉及26个生物学功能,14个通路,推测Clp X、Glm S和Cps IVK可能在两株菌致病性差异中发挥重要作用。本研究为阐明无乳链球菌不同宿主来源株致病性差异奠定了基础。

Zebrafish and macrophages were used as in vivo and in vitro infection models to evaluate the pathogenetic characteristics of Streptococcus agalactiae piscine strain（GD201008-001） and human strain（A909）. iTRAQ and LC-MS/MS technology was used to identify differentially expressed proteins in GD201008-001 and A909,and provide new clues for exploring the pathogenic mechanism of S. galactiae strains isolated from different hosts.The pathogenetic characteristics of GD201008-001 and A909 were evaluated by analyzing LD50 to zebrafish and phagocytosis of macrophages. Cellular proteins were extracted from cultures of GD201008-001 and A909, and labelled with iTRAQ reagent. Differentially expressed proteins were identified with LC-MS/MS. The mass spectrometry data was analyzed by Mascot2.2 and Proteome Discoverer1.4, and then subjected to biological information analysis of GO（gene ontology） and KEGG pathway. The result of pathogenicity difference analysis indicated that the virulence of GD201008-001 was higher than that of A909. Differentially expressed proteins were identified in S. galactiae piscine strain compared with human strain. A total of 368 differentially expressed proteins were revealed（P〈0.05）, among which 193 proteins were up-regulated（ratio〉1.5） and 175 proteins were down-regulated（ratio〈0.667） in GD201008-001. Bioinformatics analysis predicted that these proteins are mainly involved in 26 biological functions and 14 pathways. It is speculated that Clp X, Glm S and Cps IVK may play an important role in the pathogenicity of GD201008-001 and A909. These results may shed light on the pathogenic mechanism of S.galactiae strains isolated from different hosts.