Proteomic analysis of bovine mammary epithelial cells after in vitro incubation with S. agalactiae: potential biomarkers

Streptococcus agalactiae is one of the causative agents of subclinical mastitis, a common disease of dairy cows that causes great economic losses in the industry worldwide. It is thought that pathology is mainly due to infammatory damage of bovine mammary epithelial cells (bMECs); however, the mechanism by which S. agalactiae damages the bMECs is not clear. The aim of this study was to evaluate the infammatory efects of S. agalactiae on bMECs and the resulting changes in protein profles. The bMECs were incubated with S. agalactiae for diferent times and assayed for cell viability by MTT assay, apoptosis by annexin V and propidium iodide dual staining, and morphological and ultrastructural changes by scanning and transmission electron microscopy. Quantitative real-time PCR was used to determine the efect of S. agalactiae on expression of mRNA of infammatory factors in bMECs and protein levels were quantitated by liquid chromatography/mass spectrometry. Exposure to S. agalactiae signifcantly decreased the cell viability and triggered apoptosis, as well as up-regulating TNF-α, IL-1β and IL-6 mRNA, and inhibiting IL-8 expression. S. agalactiae also induced morphological and ultrastructural changes. Furthermore, we identifed 325 up-regulated and 704 down-regulated proteins in the treated vs control group. All signifcant diferentially expressed proteins (DSEPs) were classifed into three major areas by function: biological processes, cellular components and molecular functions. These diferentially expressed proteins included enzymes and proteins associated with various metabolic processes and cellular immunity. Pathway enrichment analysis showed that eight down-regulated signaling pathways were signifcantly enriched. Exposure to even subclinical levels of S. agalactiae can lead to infammation and bMEC damage. Our data suggest some possible molecular mechanisms for the harmful efects of subclinical mastitis in dairy cows.

无乳链球菌(Streptococcus agalactiae)是奶牛亚临床乳腺炎的致病菌之一，亚临床乳腺炎作为奶牛的常见疾病，给全球乳业带来了巨额经济损失。目前学界普遍认为，该病的病理损伤主要源于牛乳腺上皮细胞(bovine mammary epithelial cells, bMECs)的炎性破坏，但无乳链球菌损伤bMECs的具体分子机制仍未阐明。本研究旨在探究无乳链球菌对bMECs的炎性效应，以及其诱导的蛋白质组谱变化。实验中将bMECs与无乳链球菌共孵育不同时长，随后通过MTT法检测细胞活力，采用膜联蛋白V(annexin V)与碘化丙啶(propidium iodide)双染色法分析细胞凋亡情况，并借助扫描电镜与透射电镜观察细胞形态及超微结构的变化。采用实时荧光定量PCR(quantitative real-time PCR)检测无乳链球菌对bMECs炎性因子mRNA表达的调控作用，并通过液相色谱-质谱联用法(liquid chromatography/mass spectrometry)定量分析细胞蛋白质水平。实验结果显示，无乳链球菌暴露可显著降低bMECs的细胞活力并诱导细胞凋亡，同时上调肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)与白细胞介素-6(IL-6)的mRNA表达，却抑制白细胞介素-8(IL-8)的转录。此外，无乳链球菌还可诱导bMECs发生形态与超微结构的改变。进一步的蛋白质组分析显示，相较于对照组，处理组中共鉴定出325个上调表达蛋白与704个下调表达蛋白。所有显著差异表达蛋白(DSEPs)可按功能划分为三大类别：生物过程、细胞组分与分子功能。这些差异表达蛋白涵盖了参与各类代谢过程与细胞免疫应答的酶类及功能蛋白。通路富集分析结果显示，共有8条信号通路在下调表达蛋白中显著富集。即便是亚临床浓度的无乳链球菌暴露，也可引发炎性反应与bMECs损伤。本研究结果为解析奶牛亚临床乳腺炎的致病有害效应提供了潜在的分子机制。