猪链球菌抗氧化应激蛋白YED和AHH的鉴定

为揭示猪链球菌抗氧化应激的机制,采用比较蛋白组学方法,鉴定猪链球菌抗氧化应激相关蛋白。提取猪链球菌全菌蛋白,以THB培养基培养的细菌作为对照,经H2O2处理后,液相色谱-串联质谱共鉴定出54个差异表达蛋白,其中28个蛋白在H2O2处理组中上调表达,26个蛋白下调表达。这些差异表达的蛋白主要参与代谢、生物合成、抗应激等过程。深入分析,发现7个新的蛋白可能参与猪链球菌抗氧化应激;从中选择在H2O2处理组显著上调表达的2个蛋白YbaB/EbfC家族核苷相关蛋白(YED)和2-氨基-4-羟基-6-羟甲基二氢蝶啶二磷酸激酶(AHH)进行验证。分别构建二者缺失株ΔYED和ΔAHH;生长曲线分析表明,2个缺失株在THB培养基中生长与野生株相似;氧化应激试验表明,经H2O2作用后,野生株存活率为97.12%,而ΔYED与ΔAHH则显著降低,分别为72.69%(P<0.05)和79.49%(P<0.05);小鼠巨噬细胞RAW264.7吞噬与存活试验表明,RAW264.7对野生株和2个缺失株的吞噬率无显著差异,但与野生株相比,2个缺失株在RAW264.7内的存活率均显著降低。本研究鉴定的与氧化应激相关的差异表达蛋白,有助于揭示猪链球菌的抗氧化应激机制,其中YED和AHH与猪链球菌在吞噬细胞内存活相关,提示它们在猪链球菌致病过程中发挥作用。

Identification of YED and AHH proteins of Streptococcus suis involved in oxidative stress response

In order to reveal the mechanism of oxidative stress response in Streptococcus suis,a comparative proteomic method was used to identify the involved proteins.After H2O2 treatment,54 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry from the whole-cell extract,of which 28 were up-regulated and 26 were down-regulated in the treatment group compared to the control grown in the THB medium.These differentially expressed proteins were mainly involved in processes such as metabolism,biosynthesis,and stress response.Seven new proteins were identified,which might have been involved in oxidative stress response in S.suis.Two of the proteins,the YbaB/EbfC family nucleoside-related protein(YED)and 2-amino-4-hydroxy-6-hydroxymethyl dihydropteridine diphosphate kinase(AHH)that were significantly up-regulated in the H2O2 treatment group,were selected for verification.The deletion mutants(ΔYED andΔAHH)of the proteins were constructed.The Growth curve analysis showed that the two mutants grew in a similar manner to that of the wild-type strain in the THB medium.After H2O2treatment,the survival rate of the wild-type strain was significantly higher than that of the two mutants,which was 97.12%(the wild-type strain),72.69%(ΔYED),and 79.49%(ΔAHH),respectively.The phagocytosis and survival assay showed that,after 1 h of phagocytosis,the uptake of the wild-type strain by RAW264.7 macrophages was not significantly different from that of any of the mutant strains.However,the survival rate of the two mutants was significantly lower than that of the wild-type strain.In this study,the differentially expressed proteins identified under H2O2 stress were helpful in revealing the mechanism of oxidative stress response in S.suis,of which YED and AHH contributed to the survival of S.suis in macrophages,suggesting that they played a role in pathogenesis of S.suis.